Operator Course Workbook BD FACSCalibur BD

Operator Course Workbook
BD FACSCalibur
BD FACSort
BD FACScan
http://www.bdbiosciences.com/
337452 Rev. A
December 2003
BD Biosciences
2350 Qume Drive
San Jose, CA 95131-1807
USA
Tel (877) 232-8995
Fax (408) 954-2347
Asia Pacific
Tel (65) 6-861-0633
Fax (65) 6-860-1590
Brazil
Tel (55) 11-5185-9995
Fax (55) 11-5185-9895
Canada
Tel (888) 259-0187
(905) 542-8028
Fax (905) 542-9391
canada@bd.com
Europe
Tel (32) 53-720211
Fax (32) 53-720450
Japan
Nippon Becton Dickinson Company, Ltd.
Tel 0120-8555-90
Mexico
Tel (52) 55 5999 8296
Fax (52) 55 5999 8288
© 2003, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in
retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical,
chemical, manual, or otherwise, without prior written permission from BD Biosciences.
The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to
incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences
assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences
welcomes customer input on corrections and suggestions for improvement.
BD, the BD logo, BD Attractors, BD CaliBRITE, BD FACS, BD FACSCalibur, BD FACSFlow, BD FACSort, BD FACScan, BD FACSComp,
BD CellQuest, BD Loader Manager, BD MultiSET, BD MultiTEST, BD Worklist Manager, BD TruCOUNT, and BD Falcon are trademarks of Becton,
Dickinson and Company.
Apple Macintosh and Power Macintosh are trademarks of Apple Computer, Inc, registered in the US and other countries. Finder is a trademark of
Apple Computer, Inc.
Microsoft is a trademark of Microsoft Corporation.
Attractors software is licensed under US Patent No. 5,627,040.
PerCP is licensed under US Patent No. 4,876,190.
Clorox is a registered trademark of The Clorox Company.
All other company and product names might be trademarks of the respective companies with which they are associated.
History
Revision
Date
Change Made
Rev. A
12/03
Initial release
Contents
About This Manual
9
Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9
Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
Course Objectives
11
Chapter 1: BD FACSCalibur System
13
Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
System Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
Filling the Sheath Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
Emptying the Waste Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
System Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
Cleaning with 10% Bleach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
Rinsing with DI Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
Periodic Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Monthly Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Chapter 2: BD FACStation Data Management System
27
BD FACStation Data Management System Components . . . . . . . . . . . . . . .
28
Standard Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28
Standard Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
Optional Peripherals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
Fundamentals of the BD FACStation Data Management Filing Structure . . .
29
Types of FACStation Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
Organizing your Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
31
Useful Macintosh Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
34
Desktop Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
34
Dock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
Finder Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
38
Keyboard Shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
39
Setting the Ownership of a Folder or Document . . . . . . . . . . . . . . . . . .
40
Locking and Using Documents as Templates . . . . . . . . . . . . . . . . . . . . .
42
Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
Table of Contents
337452 Rev. A
3
Creating Folders and Organizing Files . . . . . . . . . . . . . . . . . . . . . . . . . .
43
Making an Alias on the Desktop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
44
Using the Student Folder Alias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
Optional Storage Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
46
The Basics of Working with Optional Storage Devices . . . . . . . . . . . . . .
46
Chapter 3: BD FACSComp Software
47
Performing QC and Instrument Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
Preparing BD CaliBRITE Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
48
Entering Information in the Software . . . . . . . . . . . . . . . . . . . . . . . . . . .
49
Starting a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
55
Printing and Reviewing the Summary Report . . . . . . . . . . . . . . . . . . . . .
60
Maintaining a Log of QC Results (Optional) . . . . . . . . . . . . . . . . . . . . .
61
Performing Instrument QC and Setup with the BD FACS Loader Option . . .
62
Preparing Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
Entering Information in BD FACSComp Software . . . . . . . . . . . . . . . . .
63
Setting Loader Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
65
Loading Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66
Running Beads in Walkaway Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . .
67
Reviewing Reports and Maintaining a QC Log . . . . . . . . . . . . . . . . . . .
72
Using Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
Chapter 4: BD CellQuest Pro Software
75
Optimizing Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76
Setting Up an Experiment Document . . . . . . . . . . . . . . . . . . . . . . . . . . .
77
Optimizing Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
Saving an Experiment Document . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
88
Saving Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
89
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
91
Specifying Data Collection Information . . . . . . . . . . . . . . . . . . . . . . . . .
91
Saving Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
103
Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
104
Displaying Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
105
Creating Gates and Displaying Statistics . . . . . . . . . . . . . . . . . . . . . . . .
107
Using the Expression Editor (Optional) . . . . . . . . . . . . . . . . . . . . . . . . .
113
Batch Analysis of the Remaining Data Files . . . . . . . . . . . . . . . . . . . . . .
115
Reviewing Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
Saving an Experiment Document as a Template . . . . . . . . . . . . . . . . . . . . . .
118
Importing Statistics into a Spreadsheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119
Table of Contents
337452 Rev. A
4
Using Regions and Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
120
Creating Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
120
Altering a Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
121
Using Snap-To Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
Using Region Stats to Analyze Data . . . . . . . . . . . . . . . . . . . . . . . . . . . .
128
Using Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
129
Chapter 5: BD MultiSET Software
141
Performing QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
143
Sign In View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
143
Set Up View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
144
BD FACSComp Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
145
Optimizing Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
146
Optimizing SSC Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
146
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
149
Specifying Data Collection Information . . . . . . . . . . . . . . . . . . . . . . . . .
149
Test Prefs View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
149
Samples View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
151
Sample Acquisition and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
153
Reviewing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
158
Acquiring the Remaining Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
159
Using MultiSET Tools and Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
160
MultiSET Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
160
Using User-Defined Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
165
Using Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
166
Chapter 6: DNA
167
Overview of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
168
Review of the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
168
Factors Affecting DNA Analysis by Flow Cytometry . . . . . . . . . . . . . . .
169
Standardization Requirements for DNA Flow Cytometry . . . . . . . . . . .
170
DNA Quality Control Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
DNA Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
172
Performing Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
172
Optimizing Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
ModFit LT Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
178
ModFit LT Software Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
179
DNA Analysis with ModFit LT Software . . . . . . . . . . . . . . . . . . . . . . . . . . .
180
Table of Contents
337452 Rev. A
5
Performing Auto Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
180
Performing a Manual Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
188
Analyzing Your Acquired Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
195
Creating a Database with ModFit LT Software . . . . . . . . . . . . . . . . . . . . . .
203
Setting Up the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
203
Generating the Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208
Viewing the Results in Microsoft Excel . . . . . . . . . . . . . . . . . . . . . . . . .
210
Setting S-Phase Cutoffs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
210
Analyzing Data with S-Phase Cutoffs Enabled . . . . . . . . . . . . . . . . . . . .
213
Multiparameter Gating Using ModFit LT Software . . . . . . . . . . . . . . . . . . .
214
Displaying Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
214
Gating Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
216
Locating the Diploid Channel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
218
Analyzing the Tumor Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
219
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
220
Chapter 7: Sorting
221
Sorting Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
222
Collection Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
222
Sort Line Purge Button . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
223
Sort Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
224
Sample Concentration and Selection of the Sort Mode . . . . . . . . . . . . . .
225
Effect of Event Rate on Sort Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
226
Sort Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
226
Preparing Collection Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228
Sorting Aseptically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228
Sorting Viable Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228
Sorting Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
229
Starting Up the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
229
Performing Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
230
Optimizing Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
230
Collecting a Pre-Sort Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
233
Making Selections in Sort Setup and Sort Counters . . . . . . . . . . . . . . . .
233
Sorting the Population of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
234
Concentrating the Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
235
Cleaning the Sort Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
235
Collecting a Post-Sort Data File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
237
Verifying Sort Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
237
Table of Contents
337452 Rev. A
6
Sorting Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
238
Sheath Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
238
Cell Recovery and Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
238
Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
239
Clogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
240
Testing Sorting Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
241
Ending a Sort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
242
Chapter 8: BD FACS Loader
245
Starting Up the Loader and Using It in Manual Mode . . . . . . . . . . . . . . . . .
248
Starting Up the Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
248
Installing the Rack and Loader Cover . . . . . . . . . . . . . . . . . . . . . . . . . .
249
Using the Loader Keypad Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
249
Removing the Loader Cover and Rack . . . . . . . . . . . . . . . . . . . . . . . . . .
250
Preparing Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
250
Preparing a BD CellQuest Pro Experiment Document for Use with
BD FACS Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
250
Preparing a BD MultiSET Schedule Document for Use with
BD FACS Loader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
254
Using BD CellQuest Pro Software with the Loader . . . . . . . . . . . . . . . . . . . .
257
Optimizing Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
257
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
260
Running Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
267
Reviewing the Summary Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
268
Shutting Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
269
Using BD MultiSET Software with the Loader . . . . . . . . . . . . . . . . . . . . . . .
270
Optimizing Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
270
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
272
Running Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
278
Reviewing the Summary Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
279
Shutting Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
279
Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
279
Additional Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
281
Lab Exercise: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
283
Appendix A: Theory
285
Introduction to Flow Cytometry
Compensation Theory
Table of Contents
337452 Rev. A
7
Appendix B: Worksheets
8
287
Compensation Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
288
Instrument Controls Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
289
Day 1 Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
291
Day 2 Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
294
Day 3 Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
297
DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
297
Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
298
BD MultiSET Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
300
Final Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
301
DNA Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
306
Sorting Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
307
BD FACS Loader Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
308
Troubleshooting Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
311
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
About This Manual
This training manual contains the instructions and exercises used during the operator
training course. For information about the cytometer or software not covered in this
manual, refer to the appropriate user’s guide.
Conventions
The following tables list conventions used throughout this guide.
Table 1 Notice icons
Icon

Ꮬ
;
Notice Type
Use
NOTICE
Describes important features or instructions
CAUTION
Alerts you to potential loss of data or potential damage to
an application, system, or device
WARNING
Alerts you to potential personal injury
Tip
Highlights features or hints that can save time and prevent
difficulties
Table 2 Text conventions
Convention
Use
Italics
Italics are used to highlight book titles and new or unfamiliar
terms on their first appearance in the text.
>
The arrow indicates a menu choice. For example, “choose
File > Print” means to choose Print from the File menu.
Command
Keyboard shortcuts use the Command key (a) in
combination with another indicated keystroke. For example,
Command-K means to hold down the Command key while
pressing the letter k.
About This Manual
337452 Rev. A
9
Technical Assistance
For technical questions or assistance in solving a problem, read the section of the user’s
guide specific to the operation you are performing.
If additional assistance is required, contact your local BD Biosciences technical support
representative or supplier.
When contacting BD Biosciences, have the following information available:
•
product name, part number, and serial number
•
any error messages
•
details of recent system performance
For instrument support from within the US, call (877) 232-8995, prompt 2,2.
For support from within Canada, call (888) 259-0187.
Customers outside the US and Canada, contact your local BD representative or
distributor.
Limitations
For in vitro diagnostic (IVD) use when used with IVD reagents. Refer to the
appropriate reagent package insert for application-specific limitations.
10
337452 Rev. A
Course Objectives
After completing this course, you will be able to:
•
Describe basic concepts in flow cytometry.
•
Operate and maintain the cytometer, and troubleshoot basic instrument
problems.
•
Use BD FACSComp™ software to perform quality control on the cytometer.
•
Organize data files on the Macintosh computer.
•
Acquire and analyze multicolor data using BD CellQuest™ Pro software.
If applicable, you will be able to:
•
Acquire and analyze DNA data using BD CellQuest Pro and Modfit LT software.
•
Sort cells and analyze the sorted sample to determine sort purity.
•
Use the BD FACS™ Loader option (with BD WorklistManager and
BD LoaderManager software) to acquire and analyze data.
•
Use BD MultiSET™ software to acquire and analyze data.
Course Objectives
337452 Rev. A
11
12
337452 Rev. A
1
BD FACSCalibur System
After completing this module, you will be able to:
•
Identify BD FACSCalibur™ cytometer components and how they work.
•
Perform instrument startup and shutdown procedures.
•
Describe instrument maintenance procedures.
BD FACSCalibur System
337452 Rev. A
13
The BD FACSCalibur cytometer analyzes cells as they pass one at a time through a
focused laser beam while moving in a fluid stream. The cytometer can be used for
routine research applications, immunophenotyping, and DNA cell-cycle analysis. With
the sort option, the instrument allows you to identify and sort (isolate) a subpopulation
from your sample.
The BD FACSCalibur system consists of a cytometer (Figure 1-1), computer module,
and several software packages. Many of the cytometer functions are controlled by the
software.
Cytometer
Figure 1-1 BD FACSCalibur cytometer
Power Switch
This switch, located on the bottom right side of the instrument, turns the cytometer on
and off.
Fluidics Panel
The panel where the sample flow rate and fluid modes are set
(Figure 1-2 on page 15).
14
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
fluid control buttons
sample flow rate buttons
Figure 1-2 Fluidics Control Panel
Fluidic Control Buttons
Three buttons allow you to select fluid modes.
PRIME—removes bubbles from the flow cell, used primarily during troubleshooting.
The flow of sheath fluid first stops and the pressure reverses to force fluid out of the
flow cell and into the waste tank. After a preset time, the system automatically fills the
flow cell with sheath fluid at a controlled rate to prevent bubble formation or
entrapment. After completion, the instrument goes to STANDBY mode.
RUN—increases laser to full power, starts sheath flow and pressurizes the sample tube
to transport the cell suspension through the Sample Injection Port (SIP) and into the
flow cell.
STANDBY—stops sheath flow to conserve sheath fluid and lowers laser power to
prolong laser life.
•
Hard STANDBY—When the Standby button is pressed the sheath flow stops and
the sample test tube is no longer pressurized. Additionally, the laser input current
is reduced to a standby level.
•
Auto STANDBY—If the instrument is in RUN and the tube support arm is
moved to the left or right, the instrument goes into auto standby. The RUN
button lights orange. Approximately 12 seconds after the arm is moved, the
system shuts off the sheath flow. Additionally, the laser input current is reduced to
standby level. In this mode, a vacuum pump is activated to remove sheath fluid as
it backdrips into the sample injection tube. The system returns to RUN mode
when you move the tube support arm underneath the sample tube. The sheath
flow starts and the laser returns to full power. The RUN button switches from
orange to green.
BD FACSCalibur System
337452 Rev. A
15
Sample Flow Rate Buttons
Three buttons allow you to select the sample flow rate. See Figure 1-2 on page 15.
•
LO—delivers approximately 12 µL/min of sample through the flow cell
•
MED—delivers approximately 35 µL/min of sample through the flow cell
•
HI—delivers approximately 60 µL/min of sample through the flow cell
Fluidics Drawer
The lower-left panel on the instrument that slides out for easy access to the fluid tanks
and sheath filter.
vent valve toggle switch
tab
VENT VALVE
PRESS TO
RELIEVE PRESSURE
metal bracket
J69
probe sensor connectors
J70
WASTE
SHEATH
SALINE
FILTER
sheath tank
waste tank
sheath filter
Sheath Tank
This 4-liter container, located on the left, holds enough sheath fluid for approximately
3 hours of run time. It is equipped with a fluid level detector that indicates, via the
software, a near-empty condition.
Metal Bracket
A tab on the right side of the bracket depresses a ball valve when the bracket is in the
locked position, allowing pressurization of the tank. The bracket prevents overinflation
of the tank when it is pressurized.
16
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Waste Tank
This 4-liter container, located on the right, collects the fluid waste. A fluid level detector
indicates, via the software, a near-full condition.
CAUTION
If potentially biohazardous materials are run through the BD FACSCalibur
instrument, treat this material as biohazardous waste.
Sheath Filter
This 0.22-µm filter cleans the sheath fluid before it enters the flow cell. This reduces the
amount of debris in the sheath fluid.
Vent Valve Toggle Switch
A switch that, when set in the direction of the arrow, relieves the sheath tank of air
pressure. This allows for the removal of the tank when refilling. When the switch is in
the opposite direction of the arrow, the tank pressurizes if the metal bracket is in the
locked position.
Air Filter
This component filters the air that cools the laser.
Sample Injection Port (SIP)
The SIP is the area where the sample tube is installed. It consists of the sample injection
tube through which the sample travels to the flow cell, the droplet containment system,
and the tube support arm.
Sample Injection Tube
The stainless steel tube that carries the cells from the sample tube to the flow cell. The
tube is covered with an outer sleeve that serves as part of a droplet containment system.
The droplet containment system employs a vacuum pump to aspirate fluid and prevent
drips from splashing onto the countertop.
BD FACSCalibur System
337452 Rev. A
17
sample injection tube
droplet containment tube
vacuum to waste
droplet containment
system
Figure 1-3 Droplet containment system
Tube Support Arm
The support arm’s main function is to activate the droplet containment vacuum. This
arm also supports the sample tube. The arm has three positions: centered below the
sample tube, to the right, and to the left of the sample tube. The vacuum is activated
when the arm is in the right or left position and off when the arm is centered.
Droplet Containment System
This system consists of a vacuum pump and an outer tube that surrounds the sample
injection tube. When the tube support arm is on either side of the sample injection tube,
the vacuum pump is activated, removing sheath as it backflushes from the sample
injection tube (Figure 1-3). When the tube support arm is centered, the pump is
deactivated.
CAUTION
When placing a tube on the SIP, be sure to center the tube support arm as soon
as possible to avoid having the sample aspirated and removed to the waste tank.
18
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Collection Station (Sorting Option)
The area that houses the collection tubes.
purge button
Collection Tubes
The collection tubes are located in the collection station. Three 50-mL conical tubes can
be installed in collection station. The conical tubes are used to collect the cells after they
are sorted.
Sort Lines
The sort lines are tubings that deliver the sorted sample to the collection station. The
lines are not visible from the collection station; however, periodic cleaning involves
flushing these tubings.
Sort Line Purge Button
The purge button located in the collection station automatically rinses the sort line
when pressed.
BD FACSCalibur System
337452 Rev. A
19
System Startup
Acquisition of flow cytometry data is a multistep process. The following figure outlines
the main steps involved. In this exercise, we will focus on starting up the cytometer and
the computer.
Perform
QC
Start Up
System
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Always turn on the cytometer before turning on the computer when acquiring data.
This enables the computer to recognize that the cytometer is connected. When
analyzing data, it is not necessary to turn on the cytometer.
NOTICE
An example of a daily maintenance log is provided at the end of this module.
1 Turn on the cytometer.
The power switch is located in the lower right corner of the instrument. The LO
fluid control button lights green and the STNDBY button lights orange.
2 Turn on the computer.
3 Choose your user ID and enter the password.
Filling the Sheath Tank
Recommended Sheath Fluid
•
BD FACSFlow™ sheath fluid (BD Biosciences) for nonsorting applications.
•
Phosphate-buffered saline (PBS) (Dulbecco’s Ca2+ and Mg2+-free) for sorting
applications.
Non-recommended Sheath Fluids
•
Fisher Hematology Diluent
•
Isoton III
•
Isolac D
•
Deionized (DI) water
NOTICE If you make your own sheath fluid in the lab, be sure to pass it through a
0.22-µm filter before running it on the instrument.
20
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
1 Slide the fluidics drawer out. Flip the vent valve toggle switch in the direction of
the arrow.
The switch, located between the sheath and waste tanks, relieves the sheath tank
of air pressure.
2 Disconnect the sheath tubing (white) and the air tubing (blue) from the
instrument by squeezing the metal clip on the quick-disconnect and pulling each
connector from the fitting.
3 Disconnect the fluid detection probe connector by squeezing the sides while
pulling the plastic connector.
4 Slide the metal bracket away from you, and lift up to remove it.
5 Remove the sheath tank, remove the fluid detection probe, and fill the tank to 3/4
its capacity with the recommended sheath fluid.
CAUTION
Avoid filling the sheath tank to its maximum capacity. When a full tank
is pressurized, fluid may be forced into the air supply tubing, preventing proper
pressurization. Additionally, since there is approximately 400 mL of bleach in the
4-L waste tank, filling the sheath tank to maximum capacity will overfill the waste
tank.
6 Replace the sheath tank.
7 Replace the sheath bracket by sliding the metal bracket toward you. Make sure
the square lip on the right side of the bracket is covering the ball valve.
8 Return the connectors to their original positions.
•
Insert the sheath fluid detection probe connector into its housing.
•
Insert the blue quick-disconnect into the blue port until you hear a click.
•
Insert the white quick-disconnect into the white port until you hear a click.
•
Verify there are no kinks in the sheath and waste tubing lines.
9 Flip the vent valve toggle switch to pressurize the tank.
Check the sheath tank to make sure it is properly pressurized; it should not be
able to move around under the bracket.
BD FACSCalibur System
337452 Rev. A
21
Emptying the Waste Tank
1 Disconnect the waste tubing (orange) and the air tubing (white) from the
instrument by squeezing the metal clip on the quick-disconnect and pulling.
CAUTION
It is good practice to empty the waste tank when you fill the sheath tank.
This prevents the waste tank from overflowing. Follow good laboratory practice:
wear appropriate safety attire and gloves when handling waste materials.
2 Disconnect the fluid detection probe connector by squeezing the sides and pulling.
3 Remove the waste tank, remove the fluid detection probe, and empty the tank
according to local, state, and federal hazardous waste handling regulations.
4 Fill the waste container with 400 mL of undiluted household bleach.
This will make a 10% solution of bleach in the waste container once it is full.
5 Replace the waste tank.
6 Return the connectors to their original positions.
•
Insert the waste fluid detection probe connector into its housing.
•
Insert the orange quick-disconnect into the orange-colored port until you
hear a click.
•
Insert the white quick-disconnect into the white-colored port until you hear a
click.
•
Verify there are no kinks in the waste and air vent tubing lines.
7 Check the sheath filter to be sure no air bubbles are trapped inside.
If bubbles are visible, gently tap the filter body to dislodge them and force them to
the top. Push the roller in the roller clamp forward to allow the pressurized
sheath fluid to force the air bubbles into the waste tank. Return the roller clamp
to closed position. Repeat steps if necessary.
8 Check the sheath line and the sheath filter line for bubbles.
If bubbles are in either line, disconnect the tubing at the quick-disconnect port
and press the tip of the valve against the side of a waste beaker. The pressurized
sheath fluid will force bubbles and sheath fluid out of the tubing.
NOTICE Allow the laser to warm up for 5 minutes after turning on the instrument,
before running samples.
22
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
System Shutdown
Acquisition of flow cytometry data is a multistep process. The following figure outlines
the main steps involved. In this exercise, we will focus on shutting down the cytometer
and the computer.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Always clean the instrument before you power it off at the end of the day. Proper
cleaning will ensure that your instrument will function consistently.
To prevent the sample tube from becoming clogged and to remove adhesive dyes that
could remain in the tubing causing carryover, run a bleach solution through the SIP at
the end of each day, followed by a distilled water rinse. Follow this procedure
immediately after running viscous samples or dyes such as propidium iodide (PI),
acridine orange (AO), or thiazole orange (TO).
Cleaning with 10% Bleach
1 Set the fluid control to RUN, install a tube containing 3 mL of a bleach solution
on the SIP with the support arm to the side (vacuum is on), and let it run for
1 minute.
Use BD FACS clean solution or a 1:10 dilution of bleach in DI water as the bleach
solution.
BD FACS clean solution is a bleach-based cleaning agent for daily use in
cytometer maintenance.
2 Move the support arm under the tube (vacuum is off). Allow the bleach solution
to run for 5 minutes on HI.
Rinsing with DI Water
1 Install a tube containing 3 mL of distilled water on the SIP with the support arm
to the side (vacuum is on). Let it run for 1 minute.
2 Move the support arm under the tube (vacuum is off). Allow the water to run for
5 minutes on HI.
3 Set the fluid control to STNDBY.
BD FACSCalibur System
337452 Rev. A
23
4 Place a tube containing no more than 1 mL of distilled water on the SIP.
CAUTION
Sheath fluid will backflush into the tube and may cause the tube to
overfill if more than 1 mL of distilled water is in the sample tube. This could affect
instrument performance.
5 If you are finished running samples, choose Apple menu > Shutdown to turn off
the computer, and then shut off the power to the cytometer.
The tube of distilled water should remain on the SIP to prevent salt deposits from
forming in the injection tube.
Periodic Maintenance
The following periodic maintenance procedures are necessary to maintain optimal
instrument performance.
•
Monthly cleaning
•
Sheath filter replacement
•
Air filter cleaning
•
Bal seal replacement
•
Sample O-ring replacement
The following section describes the monthly cleaning procedure. Other maintenance
procedures can be found in the FACSCalibur System User’s Guide.
Monthly Cleaning
Perform overall system fluidics cleaning at least once a month or more frequently if you
are running high volumes of samples or dyes such as propidium iodide (PI), acridine
orange (AO), or thiazole orange (TO).
1 Turn on the cytometer.
2 Remove the sheath tank.
3 Disconnect the upper tubing of the sheath filter from the SALINE FILTER port by
squeezing the metal clip on the quick-disconnect and pulling the connector from
the fitting.
4 Connect the sheath tubing (white) from the tank to the port labeled SALINE
FILTER. See Figure 1-4 on page 25.
24
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
This bypasses the sheath filter and allows fluid to travel from the sheath tank
through the tubing directly to the flow cell and to the waste tank.
VENT VALVE
PRESS TO RELIEVE
PRESSURE
J69
J70
WASTE
SHEATH
SALINE
FILTER
Figure 1-4 Bypassing the sheath filter
CAUTION
Running bleach or detergent through the sheath filter will break down
the filter paper within the filter body. These paper fragments might clog the flow
cell and require replacement of the filter.
5 Install a spare tank with 1 to 2 L of a bleach solution.
Use BD FACS clean solution or a 1:10 dilution of bleach in DI water as the bleach
solution.
6 Press the HI sample flow rate button, then install a tube containing 3 mL of
bleach solution on the SIP.
7 Press the RUN fluid control button and allow the solution to run for 20 to 30
minutes.
8 Remove the tube of bleach solution from the SIP.
OPTIONAL: Repeat steps 5 through 8 using BD FACS™ rinse solution.
BD FACS rinse solution is a detergent-based cleaning agent.
9 Repeat steps 5 through 8 using DI water.
Replace the tank containing the bleach solution with a spare tank containing 1 to
2 L of DI water. Replace the tube on the SIP with a tube containing 3 mL of DI
water.
10 Remove the tube of DI water from the SIP.
11 Replace the original sheath tank and connect the sheath filter. Place a tube
containing 1 mL of distilled water on the SIP.
BD FACSCalibur System
337452 Rev. A
25
12 Press the STNDBY button.
At this point, you can turn off the power to the cytometer if you are finished running
samples. The tube of distilled water should remain on the SIP to prevent salt deposits
from forming in the injection tube.
NOTICE You will need to run sheath fluid for approximately 5 minutes before
acquiring samples. If you will not be using your instrument for a week or longer,
perform monthly maintenance and keep the distilled water in the fluidic system until
you use the instrument again.
Contrad™ 70 is an excellent solution for removing clogs and wetting the fluidics. A
2% solution of Contrad 70 may be substituted for 10% bleach if necessary. Contrad 70
is available for purchase from BD Biosciences, cat. no. 99-30109-00.
26
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Date
Comments
Shutdown Procedure
Time Off/
Operator Initials
Max Laser Hours
(=Time off–Time on)
Monthly
Maintenance/Initials
DNA QC
HLA-B27 Calibration
Lyse/no-wash
Lyse/wash
FACSComp Pass/Fail
Startup Procedure
Date
Time On/
Operator Initials
Instrument Serial Number:
Date
Month:
Comments
CYTOMETER DAILY MAINTENANCE LOG
337452 Rev. A
2
BD FACStation Data
Management System
After completing this module, you will be able to:
•
Identify the BD FACStation data management system components
•
Describe file types, organize files, and use basic Macintosh features
•
Use and maintain optional storage devices
BD FACStation Data Management System
337452 Rev. A
27
The BD FACStationTM data management system is an Apple® Macintosh®–based
computer system, with dedicated software for acquisition, display, analysis, and storage
of flow cytometric data. Your data management system should already be installed by
your Field Service Engineer. If you need additional information on how to set up your
Macintosh computer, refer to the Getting Started manual that came with your system.
NOTICE If you need additional information on how to set up your Macintosh
computer, refer to the Macintosh user’s guide. If you are new to Macintosh, complete
the instructions provided in the pretraining packet sent by BD Biosciences before you
come to class.
This module introduces you to the components of the data management system,
explains the different types of files and how to organize them, and provides you with
helpful Macintosh features. This module also provides you basic information about
optional storage devices. For more detailed information on using the Macintosh, refer
to the Macintosh built-in online help or Apple training center (http://train.apple.com).
BD FACStation Data Management System
Components
Dedicated hardware and software are included with the BD FACStation data
management system. Optional software and peripherals can be purchased to enhance
the BD FACS system.
Standard Hardware
The standard data management system consists of the following hardware:
•
An Apple Macintosh computer
•
Color monitor
•
Keyboard
•
Mouse
•
Printer
•
Security module
The security module must be connected to your computer for you to use
BD CellQuestTM Pro software. Consult the BD CellQuest Pro Software User’s
Guide for proper attachment of the security module to your computer.
28
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Standard Software
The following software is included with the BD FACStation data management system.
•
BD CellQuest Pro software—a general purpose data acquisition and analysis
software.
•
BD FACSCompTM software—a quality control software for the cytometer.
•
BD FACSConvert software—a software that converts Flow Cytometry Standard
(FCS) 1.0 files to FCS 2.0.
Optional Peripherals
Check with your BD Biosciences Sales Representative for the availability of optional
peripherals.
Fundamentals of the BD FACStation Data
Management Filing Structure
This section was developed to help you understand how to establish your own filing
structure. To do that, there are a few fundamentals you need to understand.
This is an icon of a folder. A folder is a place to put documents. Folders allow you to
organize your filing. You should think of a folder as having the same function as a
folder in your filing cabinet. You can also put folders in other folders, to group related
subjects.
Types of FACStation Files
Various types of files and documents can be placed in folders. Following is a list of types
of files and documents along with examples of icons you might see as you use the BD
FACStation data management system.
BD FACStation Data Management System
337452 Rev. A
29
Data Files
A data file consists of unprocessed ‘raw’ data. It contains all
measured parameters for each particle (event) in a sample, as
well as information describing the sample. Data files created by
BD FACStation software are in a Flow Cytometry Standard
(FCS) 2.0 format. FACStation software programs can analyze
BD FCS 2.0 files.
Data files contain instrument settings used at the time data was
collected. You can retrieve instrument settings from FCS files and
use them to collect data, if needed.
NOTICE If you have FCS 1.0 files, you can convert to FCS 2.0 using BD FACSConvert
software. Refer to the BD FACSConvert Software User’s Guide for details.
Export Stats Files
An export stats file is a TEXT file (numbers and letters) that can
be used to transfer data obtained from an analysis into other
applications such as spreadsheet programs or database
programs. At the left are examples of two export statistics file
icons, one from BD CellQuest Pro software and one from
BD MultiSET software.
Report Files
A report file is one that contains the results of a single test or
group of tests in an organized form. Typically, report files for a
single test are PDF files. At the left are examples of two report
icons (Laboratory and Physician Reports) from
BD MultiSET software.
30
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Document files
Documents contain information entered while using the
software. The type of information stored in these files can
include plot formats, page layout, statistical markers, and
acquisition setup options. Once saved, documents can be
opened and used again at a later time. This saves the user the
time it takes to set up software options each time the program is
used. The figures on the left show an example of a
BD CellQuest Pro software Experiment document icon and a
BD MultiSET software schedule document icon.
Instrument Settings Files
An instrument settings file is one that contains all the
information necessary to set up the electronics of your cytometer
for a given application. Once saved, these settings can be recalled
from the file and sent to the cytometer. All BD data acquisition
software programs allow you to create instrument settings files.
Organizing your Files
You can organize files based on one or more of the following:
•
topics (research studies or lab applications) as shown in Figure 2-1 on page 32.
•
date (the month and date created) as shown in Figure 2-2 on page 32.
•
BD files (default for BD software) as shown in Figure 2-3 on page 33.
•
the Mac OSX default Home directory as shown in Figure 2-4 on page 33.
To help you establish a filing structure just right for your application, review the
following examples. Note that each figure displays the Finder window in Column view.
NOTICE Computers provided by BD contain two partitions, one titled Data and
another titled FACStation. The Data partition is empty. If you organize your data by
topics or date, you can save your data on the Data partition or the FACStation
partition. The FACStation partition contains all other files and applications. When you
save data using the default BD locations or the Home directory, data is saved to the
FACStation partition.
BD FACStation Data Management System
337452 Rev. A
31
Figure 2-1 Example of filing structure by topic
The filing structure in Figure 2-1 is an example of file organization by topic or study.
Folders are organized first by study, and then by month and date. It is easy to locate a
particular study by the unique name given. The example shown uses year_month_date
(030705), the recommended format when you want to sort folders by year. The month
folder can easily be archived by dragging it to a storage device icon.
Figure 2-2 Example of filing structure by date
The filing structure in Figure 2-2 is an example of file organization by date created.
Folders are organized first by month, and then by dates within the month. It is easy to
locate a particular experiment by date. Again, the example shown uses
year_month_date (030705), the recommended format when you want to sort folders by
year.
32
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 2-3 Example of filing structure by BD files
Figure 2-3 is an example file organization by software applications. Folders are
organized first by software application (FACSComp Files), and then by dated folders.
Figure 2-4 Example of filing structure by Mac OS X default
The filing structure in Figure 2-4 is an example of file organization using the Mac OS X
default Home directory. The Home directory is a folder created by the operating system
for each user. Mac OS X is designed as a multi-user system and by default tries to keep
each user’s documents and preferences separated. Therefore, when saving or opening
data in Mac OS X the dialog window defaults to a User’s Documents folder within their
Home folder.
Folders are organized by Users, user’s Home directory (eg, ellenm), Documents, and
then folders created by the user. After opening the hard drive icon, the top level
contains folders for the BD Applications and Users.
; TIP
Click the Home icon in the Finder toolbar to quickly reach the Home folder.
Each user has access to only the items inside their own Home folder, privileges to access
other user’s Home folders have to be granted by that user.
BD FACStation Data Management System
337452 Rev. A
33
Useful Macintosh Features
Following are some basic Macintosh OS X features you might find useful to assist you
in navigating the system. For additional information about the OS X operating system,
refer to the OS X software user’s guide.
Desktop Anatomy
Figure 2-5 shows the main components of the desktop. We will briefly discuss the
following.
•
Menu Bar
•
Dock
•
Finder window
menu bar
minimize
close
hard drive icon
zoom
hide toolbar
Finder
toolbar
Dock
Figure 2-5 Mac OS X desktop displaying the Finder window in column view
34
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Menu Bar
The Menu Bar contains items shown in the following graphic. We will briefly discuss
each.
Apple Menu
The Apple pull-down menu (see Figure 2-6 on page 35) contains information about
your computer, preferences, and various shutdown commands. The next section
highlights the following items in the Apple Menu.
•
System Preferences
•
Dock
•
Force Quit
Figure 2-6 Apple menu
System Preferences
The System Preferences dialog (see Figure 2-7 on page 36) contains various Macintosh
settings. System Preferences is similar to Control Panels in other operating systems.
There are four categories in the System Preferences.
•
Personal
Preferences in this section allow you to control the general look and feel of the
computer. For example, you can control the Desktop and Dock options to suit
your needs.
•
Hardware
BD FACStation Data Management System
337452 Rev. A
35
•
Internet & Network
•
System
Preferences in this section allow you to change the settings for your computer. For
example, you can create and manage user accounts and specify the startup disk.
Figure 2-7 System Preferences
Dock
The Dock option in the Apple menu quickly accesses and changes the Dock settings.
Force Quit
Force Quit will quit an application when it crashes or freezes, without rebooting the
system. This option is similar to the Windows Task Manager or pressing Command,
Option, Escape (Cmd + Opt + Esc) in previous Macintosh systems.
Finder Menu
The Finder pull-down menu contains information about the Finder, allows you to
empty the Trash, and provides access to certain utilities. The Services option allows you
to access Utilities with Grab, for making screen captures, Mail, and TextEdit, a word
processing application similar to SimpleText in previous Macintosh systems.
36
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Edit Menu
The Edit menu provides access to various editing options, like Cut, Copy, and Paste.
View Menu
The View pull-down menu controls the look of your desktop and Finder.
Go Menu
This menu provides a way to quickly navigate to various folders and locations. It also
allows you to access servers (Go > Connect to Server).
Window Menu
The Window menu controls window size and the display of windows. This menu is
important when several windows are open at the same time. To bring a hidden window
to view, choose the Bring to Front option.
Help Menu
Use the Help menu to access all help files in the system. The easiest way to use help is to
enter a question or keyword in the Ask a Question field and press Return. Figure 2-8 on
page 38 displays the results based on the keywords file format. In this example, matches
were found in three locations: Mac Help, AppleScript Help, and BD CellQuest Pro
Help.
To display help from a specific help menu:
1 Click on the Help Center icon to display all the help files available.
2 Choose the specific application.
3 Enter a question or keyword in the Ask a Question field.
NOTICE
Most BD Applications, such as BD CellQuest Pro software, now have Help.
BD FACStation Data Management System
337452 Rev. A
37
Figure 2-8 Search results window
Dock
The Dock is usually located at the bottom of the screen. It is similar to the Taskbar and
the Quick Launch toolbar in Windows or the Apple menu in previous Macintosh
systems. Besides storing minimized windows, the Dock is where you store aliases to
launch programs. It tracks open applications (black triangle beneath the open
application’s icon) and allows you to switch from one application to another.
Apple
website
Finder
applications and utilities
divider
minimized Trash
document
You can resize the Dock by clicking and dragging the divider up or down. You can
customize the dock by adding or deleting new icons (drag and drop). In Dock
Preferences (Apple menu > Dock > Dock Preferences), you can customize Dock settings
such as Dock size, magnification, and position on the screen.
Finder Window
The Finder is the application that displays the contents of a drive or folder and
functions in the same manner as previous Macintosh systems. This is similar to Explore
in Windows operating systems. The Finder window is opened by double-clicking the
hard drive icon on the desktop or by clicking on the Finder icon in the Dock.
38
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Finder Toolbar
icon list column
access to Search
function
At the top of the Finder window is the Finder toolbar. The Finder toolbar provides
access to commonly used applications, folders, and files, provides a way to change the
Finder views, and will search for files.
•
Use the View control buttons to change the look of the window to icon, list or
column view. The Finder window in Figure 2-5 on page 34 is in the column view.
•
Click the computer icon to navigate to files on disks or peripheral storage devices.
This is similar to My Computer in Windows® operating systems.
•
Click Home to navigate to your Home folder, where you can store personal files.
Each user can have a separate Home and they can also customize the Finder
toolbar to suit their needs.
•
Click the Application button to navigate to software programs and utilities. One
key utility is the Print Center. Access the Print Center by clicking the Application
button and then go to the Utilities folder. The Print Center is where you can add
or delete printer and monitor the status of your print jobs. The Print Center is a
useful item to add to your Finder toolbar.
To customize the Finder toolbar, choose View > Customize Toolbar and drag
icons off and on the toolbar.
•
Use the Search field to locate files by entering the file name and pressing Return.
Keyboard Shortcuts
A Keyboard Shortcut is a combination of key presses that initiate a command or set an
option. Several helpful keyboard shortcuts are available in the Help menu. All BD
software programs utilize the Keyboard Shortcuts as well. You can refer to the user’s
guide or the Quick Reference Guide that comes in the inside pocket of the user’s guide.
Following are examples of useful Keyboard Shortcuts.
BD FACStation Data Management System
337452 Rev. A
39
a = the Command Key (located on either side of the space bar)
aP
Selects the Print option
aS
Selects the Save option
aO
Selects the Open option
aQ
Selects the Quit option
aI
Select the Get info option
aL
Select the Make alias option
aD
Select the Duplicate file option
aC
Select the Copy option
aV
Select the Paste option
aZ
Select the Undo option
aN
Selects the New finder window option
a shift N
Selects the Open new folder option
Setting the Ownership of a Folder or Document
In the Mac OS X system, the workstation administrator can set various ownership and
access restrictions to folders and documents from the Get Info view. This is useful for
file sharing and collaborations among multiple users and groups while maintaining
individual privacy.
The folder ownership is based on a hierarchical structure. Except for the workstation
administrator(s), folders created by individual users may not be accessed by other users
unless a shared folder is created and users are given read/write privileges.
To set the ownership of a folder or document, do the following.
1 Click once on the folder or document icon for which you want to set ownership.
The folder or document icon is highlighted.
2 Choose File > Get Info.
The Get Info dialog appears. See Figure 2-9 on page 41.
40
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
lock icon
Figure 2-9 Get Info dialog box
3 Click on the disclosure triangle next to Ownership & Permissions.
4 Unlock the settings by clicking the lock icon.
5 Choose the owner of the folder or document (the creator by default) and choose
the access privilege
•
Read & Write: can make changes
•
Read only: can read the file but not make changes
•
Write only: can put files in the drop box
•
No access: cannot see the contents of the folder, nor read, or write to the file.
6 Make choices for Group, Access, and Others.
7 Click the lock icon to lock the settings.
NOTICE The Apply to Enclosed Items command allows all items in the folder to have
the same setup. However, if a folder contains items that do not belong to you, an error
message may appear. Applications and system files are set up with specific permissions.
They need to be secure to function properly. Changing them may cause the system to
display security warnings or to stop working.
BD FACStation Data Management System
337452 Rev. A
41
Locking and Using Documents as Templates
Locking or making a document a stationery pad protects the original document from
being modified. Use this feature if a document is used repeatedly for routine work, but
you do not want the original to be modified. You can open and read a locked document
but cannot make changes to the document unless you use the Save As function.
When you open a stationery pad, a copy of the document appears. You can make
changes to the document and rename it. You can use the saved stationery pad
repeatedly as a master for similar documents, such as an acquisition setup.
To lock a document or save a document as a stationery pad, do the following.
1 Click the icon of the document you want to lock or make a into a stationery pad.
2 Choose File > Get Info.
3 Click the Stationery Pad checkbox to make the document a stationery pad or
click the Locked checkbox to lock the document.
4 Close the Info dialog box.
The following graphic shows BD CellQuest Pro Experiment document icons. An
Experiment document icon that is either a standard document or a stationery pad
is shown on the left. A locked document has a padlock to the left of the document
icon and looks like the icon on the right.
lock
42
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Practice
In this tutorial, you will do the following.
•
Create and organize folders and files
•
Set the folder and document access
•
Create an alias and make a document a stationery pad
Your group will create a student folder on the hard drive. Within the Student Folder,
each person in the group will create an individual folder and set the folder access to
Read & Write. You will also make an alias of your folder and put it on the desktop.
You will then create a text document, save it in the alias folder, and make the document
a stationery pad.
Creating Folders and Organizing Files
1 Double-click the hard drive icon.
The Finder appears displaying the hard drive’s contents in either the icon, list, or
column view. Icons represent folders, programs, or documents. If you choose icon
view, you can arrange the icons by Name, Date Modified or Created, Size, or
Kind. If you choose the list view, you can sort the List by Name, Date Modified,
Size, or Kind. If you choose the column view, you can see the path of a folder or
file.
2 Choose View > as Columns to view by column.
Alternatively, click on one of the choices in the View control buttons in the Finder
toolbar, or use a3 on the keyboard.
The columns from left to right show the path of a folder or document. The icons
are listed alphabetically.
3 Click Home (
) in the Finder toolbar.
4 Verify the Student Home folder is selected and then choose File > New Folder.
An untitled folder appears within the hard drive list.
5 Enter Your Name in the highlighted text field to name the folder.
BD FACStation Data Management System
337452 Rev. A
43
6 Set the access of the Your Name Folder to Read & Write by all members in the
Info dialog box.
•
Click on the folder to select it.
•
Choose File > Get Info.
•
Click the disclosure arrow in ownership and permissions.
•
Set the Group access to Read & Write.
•
Click Apply to Enclosed Items.
•
Click OK in the message box.
•
Close the Info dialog box.
7 Repeat steps 4 through 6 for each person in your group.
Making an Alias on the Desktop
The Macintosh uses aliases as shortcuts to any folder, document, disk, or application.
This is equivalent to a shortcut on a PC. When you open an alias, it links to and opens
the original item. You will make an alias of Your Name folder so that it will be easy to
locate when saving documents and data.
1 Click the Your Name Folder to highlight it.
2 Choose File > Make Alias.
A new folder appears. The new folder will have the word alias attached to the end
of the folder’s name.
3 Drag the Your Name alias folder to the desktop, then release the mouse.
The folder icon with the Your Name alias is displayed on the Desktop.
4 Repeat steps 1 through 3 for each person in your group.
44
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Using the Student Folder Alias
When you save documents or data, it is easier to choose Your Name alias on the
Desktop than going through various folder levels to look for your folder.
1 Launch the TextEdit™ application.
To get to the TextEdit application, do the following.
•
Click the Finder icon (
) in the Dock.
•
Click the Application icon in the Finder toolbar.
•
Scroll to the TextEdit application in the window.
•
Double-click the TextEdit application icon.
An untitled document appears.
2 Enter the following text in the opened document.
Student Folder practice document.
3 Choose File > Save or Save As.
4 Enter Practice Document in the Save as field.
5 Select the document path from the Where pop-up menu.
•
Click the triangle to the right of the Where pop-up menu to display the lower
portion of the dialog box.
•
Click Desktop; select Your Name alias.
click to display lower
portion of dialog box
6 Click Save.
The Practice Document is saved in your folder in the Student Folder on the hard
drive.
BD FACStation Data Management System
337452 Rev. A
45
7 Choose TextEdit > Quit TextEdit.
8 Double-click the Your Name alias on the Desktop.
The folder opens. The Practice Document is in your folder.
Optional Storage Devices
External drives provide additional file storage space. They offer the removable media
advantage of a floppy disk with the storage capacity, media longevity, and access speed
of a hard disk. The removable media is often called a disk or a cartridge.
The Basics of Working with Optional Storage Devices
When an optional storage device is connected to your computer and a disk is in the
drive, an icon will be displayed on your desktop. You can access your optional storage
device like any other disk drive. To help keep your system running smoothly remember
the following:
•
Refer to the manufacturer’s user’s guide for startup and shutdown procedures.
•
The optional storage device must be turned on before the computer, so that it can
be recognized by the computer.
•
New media must be formatted before they can be used. Refer to the
manufacturer’s user’s guide for details.
•
Before powering the system off, remove the disk from the drive. Failure to do so
may result in data loss.
•
For installation or troubleshooting information, refer to the manufacturer's user's
guide.
•
Never connect devices to or disconnect them from the computer while power to
the computer or any of the devices is on. Failure to power down the computer
and all connected devices may cause damage to the device or computer.
•
If the power fails while a disk is in the drive, do not attempt to remove the disk
until power is restored. If you must remove a disk while the drive is without
power, contact your manufacturer’s customer service.
CAUTION Never save data directly to an optional disk. The FACStation works most
efficiently when data is saved directly to your hard drive and then copied to your
optional drive. Communication problems resulting in data loss may be encountered
when saving directly to optional drives.
46
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
BD FACStation
Data Management
System
337452 Rev. A
File Types
Data files
Export stats files
Report files
Document files
Instrument settings files
2
1
File Organization
Data can be organized by:
• Topic
• Date
• Users
• BD Files
• OSX Home Directory
3
Data Management
Files Organized by OSX Home Directory
4
2
3
BD FACSComp Software
After completing this module, you will be able to:
•
Use BD FACSComp™ software with or without the BD FACS™ Loader.
•
Perform instrument quality control and set up the cytometer for
immunophenotyping of human peripheral blood.
BD FACSComp Software
337452 Rev. A
47
Acquisition of flow cytometry data is a multistep process. The following figure outlines
the main steps involved. In this module, we will focus on instrument quality control
(QC).
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Instrument QC is performed by running BD CaliBRITE™ beads with BD FACSComp
software. The software monitors instrument performance and provides automated
instrument setup. This software, when used with BD CaliBRITE beads, provides
routine instrument setup for two-, three- and four-color immunophenotyping
applications of human cells.
A calibration file is created each time BD FACSComp is run through to completion.
Calibration files contains all instrument settings as well as the overall result of the
Sensitivity Test encoded in a keyword (TRUE if all parameters pass; FALSE if any
parameter fails). Only one version of each calibration file can exist at a time. Each time
a new assay is run, the new file overwrites the existing file. Refer to the BD FACSComp
Software User’s Guide for details about the software and its functions.
Before beginning the following exercise, start up the cytometer and computer if they are
not already on. See System Startup on page 20 in the BD FACSCalibur System module
for details.
Follow the Performing Instrument QC and Setup with the BD FACS Loader Option on
page 62 if you have a cytometer equipped with the BD FACS Loader option. If you do
not have the Loader option, do the following exercise.
Performing QC and Instrument Setup
In the following exercises, you will:
•
Prepare BD CaliBRITE beads.
•
Enter information in BD FACSComp software.
•
Run three- or four-color beads.
•
Print and review the Summary Report.
Preparing BD CaliBRITE Beads
In this exercise, you will prepare three- or four-color beads.
1 Mix the stock vials of BD CaliBRITE beads to thoroughly suspend them.
Do this by gently vortexing the vials.
48
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
2 Label one 12 x 75-mm tube A and another B. Add 1 mL of BD FACSFlow™
sheath fluid to tube A. Add 3 mL to tube B.
NOTICE For calibrations using PerCP-Cy5.5 beads, prepare bead suspensions in
the bead dilution buffer provided with the beads.
3 Using Table 3-1, choose the setup that you are to run in BD FACSComp software.
Add one free-falling, complete drop of each of the BD CaliBRITE bead vials
based on the information in each column.
Table 3-1 Beads preparation table
Setup
Unlabeled
Tube A
Mixed Tube B
two-color setup
unlabeled
unlabeled, FITC, PE
three-color setup using
PerCP
unlabeled
unlabeled, FITC, PE, PerCP
three-color setup using
PerCP-Cy5.5
unlabeled
unlabeled, FITC, PE,
PerCP-Cy5.5
four-color setup
unlabeled, APC
unlabeled, FITC, PE, PerCP,
APC
four-color setup using
PerCP-Cy5.5
unlabeled, APC
unlabeled, FITC, PE,
PerCP-Cy5.5, APC
4 Cap the tubes and mix by gentle inversion.
CAUTION
If PerCP beads are included in the mixed bead suspension, beads are
stable for 1 hour at 4°C. (Bead stability at room temperature is less.)
Entering Information in the Software
In this exercise you will do the following.
•
Enter Operator name at the Sign In view.
•
Enter BD CaliBRITE beads lot IDs at the Set Up view.
1 Click the BD FACSComp software icon in the dock.
A startup screen appears, then the application window opens with the Sign In
view displayed. See Figure 3-1 on page 50.
BD FACSComp Software
337452 Rev. A
49
Figure 3-1 Sign In view
2 Fill in the Operator field.
Only the operator field is required. Verify that the Cytometer Model and Serial
Number are correct.
3 Click Accept or press Return.
The Set Up view appears. Notice under each Assay Selection the status of the last
calibration file is displayed.
50
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 Click Lyse/Wash(LW), and if appropriate, LWPerCP-Cy5.5, under Assay
Selection.
BD FACSComp software automatically determines whether two-, three-, or fourcolor setup is being performed based on the lot IDs entered and the bead
populations detected during acquisition. Up to five different assays can be selected
and run in a continuous fashion.
5 Enter each BD CaliBRITE bead lot ID as it appears.
For four-color setup, also enter the APC bead lot ID.
The bead lot ID, consisting of five numbers and one letter, is found on the sticker
included with the BD CaliBRITE kit. Do not add a space between the numbers
and letter. The suffix ID is case sensitive; type capital letters. The last character is
a code for a channel separation value. For unlabeled beads, the last character
encodes FSC and SSC minimum separation values. For fluorescent labeled beads,
the last character encodes FL1, FL2, FL3, and FL4 minimum separation values.
These values are used to evaluate instrument performance during the Sensitivity
Test. The APC bead lot ID also provides the FL4 PMT target channel value.
6 Enter an FSC Threshold and Gain, if needed.
NOTICE If you know your application’s usual threshold value (and gain, if
applicable), you can enter it here. The values you enter are not used by
BD FACSComp software in setting up the cytometer, but are added to the
appropriate Calib File. When the Calib File settings are downloaded to the
cytometer, these values are also downloaded to facilitate setup. Refer to the
BD FACSComp Software User’s Guide for details.
7 Click the Lyse/Wash Summary Report checkbox if you want to save a summary
of the results.
The Summary Report file contains the analytical results of a single run. If you
click the Location button, a directory dialog appears, where you can specify the
name and storage location of the report. The default name of the Summary
Report is DDMMYY.sum for the text file and DDMMYY.sum.PICT for the pict
file. You can also change the prefix. The default location is BD Files: FACSComp
Files:DDMMYY folder.
Verifying Target Values
You will need to input target values in BD FACSComp software when instructed by BD.
Target values change when a new lot of raw bead materials is used to make
BD CaliBRITE beads. This usually occurs periodically, eg, once a year or once every
two years.
It is a good idea to verify the target values in the software are the correct values,
especially if you are in a multi-user lab. Do the following to verify target values.
BD FACSComp Software
337452 Rev. A
51
1 Choose FACSComp > Edit Target Values.
A dialog appears.
2 Click OK.
3 Compare the target values listed in the dialog that appears with those listed on
the target values insert from the BD CaliBRITE bead kit you are using.
Note that there are target values for Lyse Wash and Lyse/No-Wash assays. To
view target values for Lyse/No-Wash, select it from the Target File pop-up menu.
Target File pop-up
menu
4 Enter different values, if needed.
5 Click OK to exit the dialog and save the settings.
52
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Setting Preferences
You can change certain default settings for BD FACSComp software in the Preferences
dialog.
1 Choose FACSComp > Preferences.
The Preferences dialog appears. See Figure 3-2 on page 53.
Figure 3-2 Preferences dialog
2 Click the Export icon.
3 Select Tab.
The file format selected applies to both the Summary Report and the LJ Data file.
BD FACSComp Software
337452 Rev. A
53
4 Click the Printing icon.
5 Choose Print after each assay to automatically print a Summary Report at the
conclusion of each assay.
6 Scroll down the icon list and click the LJ Data icon.
BD FACSComp software allows you to save Summary Report results to a file that
can be exported to a third-party statistical software application.
NOTICE Make sure to select the appropriate Summary Report checkbox under the
Automatic Savings Options at the Set Up view to save Levey-Jennings files.
7 Select 30 runs.
The export file will be appended with new data until the specified number of runs
has been saved; thereafter, a first in/first out method applies.
8 Click the Loader icon.
The Loader preferences allow you to specify whether or not you will be using a
BD FACS Loader during the BD FACSComp software run. For this exercise,
make sure the Run With the Loader option is NOT selected.
9 Click Save to save all the selected preferences.
The Preferences are saved.
10 Click Run at the Set Up view.
The PMT view appears. See Figure 3-3 on page 55.
54
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 3-3 PMT view
Starting a Run
In this exercise, you will start the run and BD FACSComp will do the following.
•
Adjust PMT settings until target values are reached.
•
Perform time-delay calibration (for 4-color setup).
•
Adjust compensation settings until target values are reached.
•
Test sensitivity in all parameters and indicate in the Summary Report whether
sensitivity passed or failed.
1 Gently mix the unlabeled (or unlabeled and APC) bead tube, and install it on the
cytometer.
2 Push the RUN button on the cytometer and set the flow rate to HI.
3 Click Start to begin acquiring events.
A status message at the bottom of the PMT Adjustment view continuously
displays the % Complete for the singlet data acquisition. See Figure 3-4 on
page 56.
NOTICE For instruments with the FL4 option, the software determines whether
APC beads are present. If no APC beads are detected, the second laser is turned
off and PMTs are adjusted for two- or three-color setup. If APC beads are
detected and the APC bead lot ID was entered at the Set Up view, the second laser
remains on, the FL4 PMT is adjusted, and an FL4 histogram appears.
BD FACSComp Software
337452 Rev. A
55
Figure 3-4 PMT view with FL4 histogram
PMT Adjustments
BD FACSComp software adjusts the PMTs to place the beads in their respective target
channels.
When the PMTs are set, checkmarks appear next to the PMT labels listed below the
plots. The values next to the PMT labels are the mean channel numbers of the singlet
beads in each parameter. The message PMTs Set Successfully appears when the values
displayed are close to the assigned PMT target values. The software counts down for 5
seconds, allowing you to pause before it automatically advances to the Compensation
view for three-color compensation, or time-delay calibration, if you are performing
four-color setup.
1 If necessary, perform manual adjustments.
BD FACSComp software will go into a manual mode if either of the following
conditions are met:
-
sample flow rate is less than 400 events/sec.
-
Automatic PMT adjustments take longer than 75 seconds.
To activate the manual mode, click the Manual button at the bottom of the
screen. The Target Value window appears. Use the Detectors/Amps window to set
the PMTs to ±2 channels of the target values for the fluorescence PMTs. For SSC,
set the PMT to ±5 channels of the target value. Checkmarks do not appear during
manual PMT adjustment.
56
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Time-Delay Calibration (Four-Color Setup)
If APC beads were detected, BD FACSComp software automatically performs a timedelay calibration immediately after the PMTs are set.
Leave the unlabeled and APC beads on the cytometer until time-delay calibration is
complete.
A message appears indicating time-delay calibration is being performed.
If the calibration is successful, a message appears for 3 seconds; then the software
proceeds to the Compensation view.
If the calibration is not successful, you have 5 seconds to decide whether to retry timedelay calibration, repeat initial PMT adjustment, or continue to compensation.
If you do not select an option within 5 seconds, the software automatically advances to
the Compensation view.
CAUTION
If you continue with compensation after a failed time-delay calibration, the
compensation values you obtain for FL3–%FL4 and FL4–%FL3 might vary
substantially. A failed time-delay calibration will be noted in the comments section of the
Summary Report. If compensation does not appear to be correct during optimization,
troubleshoot for time-delay calibration errors. See the BD FACSComp Software User’s
Guide for more information.
BD FACSComp Software
337452 Rev. A
57
After the PMTs are successfully set, (and time-delay calibration is complete), the
software automatically proceeds to the Compensation view.
Compensation Adjustment
1 Remove the unlabeled (or unlabeled and APC) bead tube from the cytometer.
2 Gently mix the mixed bead tube, and install it on the cytometer.
3 Click Start to begin compensation adjustment.
BD FACSComp software adjusts the appropriate compensation values until the
target mean difference (or target mean for FL4) is achieved. If APC beads are not
detected, three-color setup is performed. If PerCP beads are not detected, twocolor setup is performed. When compensation is set, checkmarks appear and the
message Compensation Set Successfully appears. The software counts down for 5
seconds, allowing you to pause before it automatically advances to the Sensitivity
view.
CAUTION
Running the wrong FL3 beads will result in inaccurate FL4–%FL3
compensation results. Make sure the assay corresponds with the BD CaliBRITE
beads to be run.
4 If necessary, perform manual adjustments.
BD FACSComp software will go into a manual mode if any of the following
conditions are met:
58
-
Flow rate is less than 400 cells/sec.
-
Automatic compensation takes longer than 75 seconds.
-
Compensation value is greater than 50%.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
To activate the manual mode, click the Manual button at the bottom of the
screen. The Target Value dialog appears. Use the Compensation dialog to adjust
compensation manually. The mean differences are displayed below the plots.
These values should be within ±2 channels of the target values.
Sensitivity Tests
1 After compensation is successfully set, the Sensitivity Test view appears. See
Figure 3-5 on page 59.
Leave the mixed bead tube on the cytometer.
The Sensitivity Test consists of three different tests: the Fluorescence Sensitivity
Test, the Side Scatter Sensitivity Test, and the Forward Scatter Sensitivity Test.
Figure 3-5 Sensitivity Test view
Fluorescence Sensitivity Test
The software acquires 10,000 events and calculates fluorescence separation between the
means of the unlabeled and labeled beads in the FL1, FL2, and FL3 channels. FL4
sensitivity is measured by calculating the separation between the means of the PerCP
(or PerCP-Cy5.5) and APC bead signals in the FL4 channel.
Side Scatter Sensitivity Test
The cytometer produces an electronic test pulse in the FSC parameter. For
BD FACSCalibur and BD FACSort cytometers, the test pulse is automatically turned on.
For BD FACScan instruments, you will be prompted to turn on test pulses.
BD FACSComp software acquires 10,000 events, locates the upper test pulse peak
channel, then acquires data for the side scatter Sensitivity Test. The separation between
noise and the side scatter signal of the beads is calculated. When this is complete, the
test pulses are automatically turned off (BD FACSort and BD FACSCalibur flow
BD FACSComp Software
337452 Rev. A
59
cytometers), or you are prompted to turn them off (BD FACScan flow cytometer), and
the forward scatter Sensitivity Test begins.
Forward Scatter Sensitivity Test
For this test, the electronic test pulse is turned off. The cytometer produces an electronic
test pulse in the FSC parameter. For BD FACSCalibur and BD FACSort cytometers, the
test pulse is automatically turned off. For BD FACScan instruments, you will be
prompted to turn off test pulses.The software searches for the upper level of FSC noise
by lowering the FSC threshold. After it finds the noise level, the software sets the
threshold, acquires 10,000 events and calculates the separation between the threshold
and the mean channel FSC signal of the beads.
Ending a Run
1 Remove the beads and set the cytometer to standby mode.
2 Click one of the following buttons.
-
Next Assay to continue with the next assay. If this was the last assay, this
button becomes Set Up, which proceeds to the Set Up view.
-
Quit to quit BD FACSComp software.
Printing and Reviewing the Summary Report
BD FACSComp software displays the Summary Report view in a scrollable window.
See Figure 3-6 on page 61. If you selected Print After Each Assay in the Print
Preferences, the Summary Report prints automatically when you leave the Summary
view. If you selected not to print the Summary Report, you can print it while it is
displayed by selecting Print from the File menu.
The Summary Report contains histograms and the separations for each parameter, the
current instrument settings, results of the time-delay calibration (if you performed a 4color setup), and the Sensitivity Test results. If the channel separation is greater than or
equal to the minimum channel separation, the parameter will pass the Sensitivity Test.
Scroll down the window to view the Sensitivity Test histograms.
To enter comments on the report:
•
scroll down to the Comments section
•
click to insert the cursor
•
type
If you exceed three lines of text, the text flows to a new page. Comments may be added
only before the file is saved, and before leaving the Summary view.
60
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4-Color Lyse/Wash FACSComp Report
nstitution:
irector:
perator:
.Swenson
Parameter
High
Low
Separation
Minimum
FSC
163
26
137
100
Pass
59716I
SSC
237
1
236
210
Pass
59716I
Pass
59715O
FL1
a t e : hu, Aug 14, 2003 1:34 PM
oftware: ACSComp 5.1
ytometer: ACSCalibur E0053
202
75
127
esult
93
Lot ID
FL2
207
63
144
120
Pass
60122J
FL3
207
60
147
120
Pass
59713N
FL4
211
142
69
48
Pass
60524I
Parameter
Detector
Amplifier
Threshold
FSC
E00
2.00
52
SSC
FL1
339
617
1.00
Log
FL2
FL3
668
821
Log
Log
FL4
704
Log
Compensation FL1-%FL2
FL2-%FL1 FL2-%FL3
1.1
additional comments
0.0
18.2
lue Laser Current 6.50 Amps
lue Laser Power
14.95 mWatts
FL3-%FL2
FL3-%FL4
FL4-%FL3
17.9
1.7
7.0
0
50 100 150 200 250
FSC-H
0
50
100 150 200 250
FL1-H
0
50
100 150 200 250
FL3-H
0
50 100 150 200 250
SSC-H
0
50
100 150 200 250
FL2-H
0
50
100 150 200 250
FL4-H
omments:
ime Delay Calibration Passed. Lab practical
Figure 3-6 Summary Report
Maintaining a Log of QC Results (Optional)
You can maintain a log of your QC results, if needed. Do one or more of the following.
•
Print the Summary Report.
•
Save an electronic file of the Summary Report.
•
Manually enter results from the Summary Report in a QC log.
During the training week, enter values from your Summary Report into the log
provided at each computer workstation.
A copy of this log is provided at the end of this chapter for use in your lab. Enter any
notes or comments you want to document for a particular run, such as lot ID change,
instrument maintenance, or instrument problems.
BD FACSComp Software
337452 Rev. A
61
Performing Instrument QC and Setup with the
BD FACS Loader Option
BD FACSComp software version 5.1 and higher include Loader support. Refer to the
BD FACSComp Software User’s Guide or the BD FACS Loader User’s Guide for
details.
In the following exercises, you will:
•
Prepare BD CaliBRITE beads.
•
Enter information in BD FACSComp software, including Loader preferences.
•
Run three- or four-color beads using the Loader in walkaway operation.
•
Print and review the Summary Report.
Preparing Beads
In this exercise, you will prepare three- or four-color BD CaliBRITE beads.
1 Mix the stock vials of BD CaliBRITE beads to thoroughly suspend them.
Do this by gently vortexing the vials.
2 Label one 12 x 75-mm tube A and another B. Add 1 mL of BD FACSFlow™
sheath fluid to tube A. Add 3 mL to tube B.
NOTICE For calibrations using PerCP-Cy5.5 beads, prepare bead suspensions in
the bead dilution buffer provided with the beads.
3 Using Table 3-1 on page 49, choose the setup that you are to run in
BD FACSComp software. Add one free-falling, complete drop of each of the
BD CaliBRITE bead vials based on the information in each column.
4 Cap the tubes and mix by gentle inversion.
CAUTION
If PerCP beads are included in the mixed bead suspension, beads are
stable for 1 hour at 4°C. (Bead stability at room temperature is less.)
62
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Entering Information in BD FACSComp Software
In this exercise you will do the following.
•
Enter Operator, Institution, and Director Information at the Sign In view.
•
Enter BD CaliBRITE bead IDs at the Set Up view.
1 If you have not already done so, turn on the power to the Loader with the switch
on the Loader keypad.
2 Click the BD FACSComp software icon in the dock.
A startup screen appears, then the application window opens with the Sign In
view displayed.
3 Fill in the Operator field.
Only the operator field is required. Verify that the Cytometer Model and Serial
Number are correct.
4 Click Accept or press Return.
The Set Up view appears. See Figure 3-7 on page 64.
BD FACSComp Software
337452 Rev. A
63
Figure 3-7 Set Up view
5 Click the Lyse/Wash (LW) and the Lyse/No-Wash (LNW) checkboxes under Assay
Selection.
In this exercise, you will perform both assays to demonstrate the function of
running more than one assay. BD FACSComp software performs the selected
assays in the order they are listed. The software will automatically determine
whether two-, three-, or four-color setup is being performed based on the lot IDs
entered and the bead populations detected during acquisition. Up to three
different assays can be selected and run in a continuous fashion.
6 Enter each BD CaliBRITE bead lot ID as it appears.
For four-color setup, also enter the APC bead lot ID.
The bead lot ID, consisting of five numbers and one letter, is found on the flyer
included with the BD CaliBRITE kit. Do not add a space between the numbers
and letter. The suffix ID is case sensitive; type capital letters. The last character is
a code for a channel separation value. For unlabeled beads, the last character
encodes FSC and SSC minimum separation values. For fluorescent labeled beads,
the last character encodes FL1, FL2, FL3, and FL4 minimum separation values.
These values are used to evaluate instrument performance during the Sensitivity
Test. The APC bead lot ID also provides the FL4 PMT target channel value.
7 If you are running the Lyse/Wash assay, enter an FSC Threshold and Gain, if
needed. If you are running the Lyse/No-Wash assay, enter a FL3 threshold, if
needed.
NOTICE If you know your application’s usual threshold value (and gain, if
applicable), you can enter it here. The values you enter are not used by
BD FACSComp software in setting up the cytometer, but are added to the
appropriate Calib File. When the Calib File settings are downloaded to the
64
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
cytometer, these values are also downloaded to facilitate setup. Refer to the
BD FACSComp Software User’s Guide for details.
8 Click the Lyse/Wash Summary Report and Lyse/No-Wash Summary Report
checkboxes if you want to save a summary of the results.
The Summary Report file contains the analytical results of a single run. If you
click the Location button, a directory dialog appears, where you can specify the
name and storage location of the report. The default name of the Summary
Report is DDMMYY.sum for the text file and DDMMYY.sum.PICT for the pict
file. You can also change the prefix. The default location is BD Files: FACSComp
Files:DDMMYY folder.
Setting Loader Preferences
The Loader preferences allow you to select whether or not you will be using a Loader
during the run.
1 Choose FACSComp > Preferences and click the Loader icon.
A dialog appears with an option for running with the Loader.
2 Select the Run With The Loader checkbox.
3 Select the Walkaway Operation checkbox.
The software will automatically lift the appropriate tube to the SIP and advance
through PMT, time-delay calibration (four-color assays only) compensation, and
sensitivity testing for LW, then LNW assays (if chosen) without any user
intervention.
NOTICE When running walkaway operation with a BD FACScan flow cytometer,
you must be present to turn on the test pulses during the Sensitivity testing.
BD FACSComp Software
337452 Rev. A
65
4 Select the Automatic Clean checkbox, if necessary.
A 3-minute clean cycle will run automatically when you quit the software. Place
the bleach tube in position 39 and distilled water in position 40. Each tube runs
for 1-1/2 minutes.
5 Set other preferences, as needed.
See Verifying Target Values on page 51.
6 Click Save.
Loading Tubes
The following tube positions must be used when running BD FACSComp software with
the Loader. If you are not running a particular assay, leave the corresponding tube
position empty.
Position
Tube
1
Unlabeled beads
2
Mixed beads for LW and LNW not containing
PerCP-Cy5.5
3
Mixed beads for LW PerCP-Cy5.5 and LNW PerCP-Cy5.5
4
HLA-B27 calibration beads
5–38
Optimization samples
39
3 mL of 10% bleach solution or undiluted BD FACS Clean solution
40
DI water (3 mL)
1 Place the unlabeled and mixed bead tubes in the appropriate positions on the rack
according to the positions listed in the table above.
2 Place a tube containing bleach solution (3 mL) in position 39 and a tube
containing DI water (3 mL) in position 40 on the rack.
3 If necessary, remove the DI water tube from the SIP and move the support arm to
the side.
4 Place the rack on the Loader.
NOTICE Any rack may be used.
5 Push the drawer in, and put the Loader cover in place.
The Loader cycles and Tube 01 is displayed on the Loader keypad control.
66
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
6 Make sure the sample flow rate is set to HI and the cytometer is set to RUN.
7 Click Check Seal at the bottom of the Set Up view.
The rack spins and the Loader raises tube 40 to verify that a seal can be formed
by the lifter. Once the seal is verified, the tube is lowered and a dialog box
displays the results of the Check Seal test.
Running Beads in Walkaway Mode
In this exercise, you will start the run and BD FACSComp will do the following.
•
Adjust PMT settings until target values are reached.
•
Perform time-delay calibration (for 4-color setup).
•
Adjust compensation settings until target values are reached.
•
Test sensitivity in all parameters and indicate in the Summary Report whether
sensitivity passed or failed.
PMT Adjustment
1 Click Run at the Set Up view.
The PMT view appears.
The Loader lifts tube 1 to the SIP and acquisition begins.
BD FACSComp Software
337452 Rev. A
67
The window at the bottom of the PMT Adjustment view continuously displays
the % Complete for the singlet data acquisition.
NOTICE For instruments with FL4 option, the software determines whether APC
beads are present. If no beads are detected, the second laser is turned off and
PMTs are adjusted for two- or three-color setup. If APC beads are detected and
the APC bead lot ID was entered at the Set Up view, the second laser is turned on,
the FL4 PMT is adjusted, and an FL4 histogram appears.
BD FACSComp software adjusts the PMTs to place the beads in their respective
target channels.
When the PMTs are set, checkmarks appear next to the PMT labels listed below
the plots. The values next to the PMT labels are the mean channel numbers of the
singlet beads in each parameter. The message PMTs Set Successfully appears when
the values displayed are close to the assigned PMT target values. The software
counts down for 5 seconds, allowing you to pause before it automatically
advances to the Compensation view (or time-delay calibration, if you are
performing four-color setup).
2 If necessary, perform manual adjustments.
BD FACSComp software will go into a manual mode if either of the following
conditions are met:
-
Flow rate is less than 400 events/sec.
-
Automatic PMT adjustments take longer than 75 seconds.
To activate the manual mode, click the Manual button at the bottom of the
screen. The Target Value window appears. Use the Detectors/Amps window to set
the PMTs to ±2 channels of the target values for the fluorescence PMTs. For SSC,
set the PMT to ±5 channels of the target value. Checkmarks do not appear during
manual PMT adjustment.
68
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Time-Delay Calibration (Four-Color Setup)
If APC beads were detected, BD FACSComp software automatically performs a timedelay calibration immediately after the PMTs are set.
A message appears indicating time-delay calibration is being performed.
If the calibration is successful, a message appears for 3 seconds, and the software
proceeds to the Compensation view.
If the calibration is not successful, you have 5 seconds to decide whether to retry timedelay calibration, repeat initial PMT adjustment, or continue to compensation.
If you do not select an option within 5 seconds, the software automatically advances to
the Compensation view.
CAUTION
If you continue with compensation after a failed time-delay calibration, the
compensation values you obtain for FL3–%FL4 and FL4–%FL3 might vary
substantially. A failed time-delay calibration will be noted in the comments section of the
Summary Report. If compensation does not appear to be correct during optimization,
troubleshoot for time-delay calibration errors. See the BD FACSComp Software User’s
Guide for more information.
After the PMTs are successfully set, (and time-delay calibration is complete), the
software automatically proceeds to the Compensation view.
The Loader raises tube 2 to the SIP, the Compensation view appears, and acquisition
begins.
BD FACSComp Software
337452 Rev. A
69
Compensation Adjustment
BD FACSComp software adjusts the appropriate compensation values until the target
mean difference (or target mean for FL4) is achieved. If APC beads are not detected,
three-color setup is performed. If PerCP beads are not detected, two-color setup is
performed. When compensation is set, checkmarks appear and the message
Compensation Set Successfully appears. The software counts down for 5 seconds,
allowing you to pause before it automatically advances to the Sensitivity view.
3 If necessary, perform manual adjustments.
BD FACSComp software will go into a manual mode if any of the following
conditions are met:
-
Flow rate is less than 400 cells/sec.
-
Automatic compensation takes longer than 75 seconds.
-
Compensation value is greater than 50%.
To activate the manual mode, click the Manual button at the bottom of the
screen. The Target Value dialog appears. Use the Compensation dialog to adjust
compensation manually. The mean differences are displayed below the plots.
These values should be within ±2 channels of the target values.
Sensitivity Tests
After compensation is successfully set, the Sensitivity Test view appears. See Figure 3-8
on page 71. The Fluorescence Sensitivity acquisition begins.
70
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 3-8 Sensitivity Test view
The Sensitivity Test consists of three different tests: the Fluorescence Sensitivity Test, the
Side Scatter Sensitivity Test, and the Forward Scatter Sensitivity Test.
Fluorescence Sensitivity Test
The software acquires 10,000 events and calculates fluorescence separation between the
means of the unlabeled and labeled beads in the FL1, FL2, and FL3 channels. FL4
sensitivity is measured by calculating the separation between the means of the PerCP
(or PerCP-Cy5.5) and APC bead signals in the FL4 channel.
Side Scatter Sensitivity Test
The cytometer produces an electronic test pulse in the FSC parameter. BD FACSComp
software acquires 10,000 events, locates the upper test pulse peak channel, then
acquires data for the side scatter Sensitivity Test. The separation between noise and the
side scatter signal of the beads is calculated. When this is complete, the test pulses are
automatically turned off (BD FACSort and BD FACSCalibur flow cytometers), or you
are prompted to turn them off (BD FACScan flow cytometer), and the forward scatter
Sensitivity Test begins.
Forward Scatter Sensitivity Test
For this test, the electronic test pulse is turned off. The software searches for the upper
level of FSC noise by lowering the FSC threshold. After it finds the noise level, the
software sets the threshold, acquires 10,000 events and calculates the separation
between the threshold and the mean channel FSC signal of the beads.
BD FACSComp Software
337452 Rev. A
71
The Summary Report appears after the sensitivity tests are complete. See Figure 3-6 on
page 61.
The Lyse/Wash Summary Report prints automatically when you leave the Summary
view. The software counts down for 5 seconds allowing you to pause before it
automatically advances to the next assay (Lyse/No-Wash in this exercise).
If you selected to run more than one assay, the software advances to the PMT View of
the Lyse/No-Wash assay. Automatic adjustment begins, followed by time-delay
calibration (four-color setup only), compensation adjustment and sensitivity testing.
Upon completion of the last assay, the software proceeds to the Set Up view, the tube is
unloaded, and the calibration file is saved.
Ending a Run
1 Click Quit.
The automatic cleaning cycle begins. Tube 39 runs for 1 1/2 minutes followed by
tube 40, which runs for 1 1/2 minutes.
2 Remove the rack when the cleaning cycle is complete.
3 Install a tube containing no more than 1 mL of DI water on the SIP and place the
cytometer in standby.
Reviewing Reports and Maintaining a QC Log
1 See page 60 for details about printing and reviewing the Summary Report.
2 See page 61 for details about maintaining a log of your QC results.
72
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Using Help
When you need assistance with BD FACSComp software, use the Help viewer to
quickly locate the information.
1 From the Help menu, choose FACSComp Help.
The fastest way to search for help is to use the Ask a Question feature. You can
also search by looking through the table of contents or index.
2 Enter sensitivity test in the Ask a Question field; press Return.
A list of topics appears in the lower portion of the Help view. Double-click on a
topic to go to that section of Help.
BD FACSComp Software
337452 Rev. A
73
3 When you are finished, close the Help viewer by clicking the red button in the
upper-left corner of the viewer.
4 Click Quit to exit the software.
74
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
BD FACSComp™
Software
337452 Rev. A
The Big Picture
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
2
1
FACSComp — Uses
• To monitor daily cytometer performance
• To set up the cytometer for two-, three-,
and four-color immunophenotyping
applications of human cells
3
How FACSComp Works
• FACSComp adjusts the cytometer settings
to position the CaliBRITE beads in
standard target channels.
• FACSComp checks the sensitivity of the
cytometer by evaluating the channel
separation of CaliBRITE beads.
• User logs or exports the summary data
daily.
4
2
Functions of FACSComp
• PMT voltage adjustment
• Time-delay calibration
• Fluorescence compensation adjustment
• Sensitivity testing
5
Preparing the CaliBRITE Bead
Suspensions
Add 1.0 mL sheath fluid* to Tube A and 3.0 mL
sheath fluid to Tube B.
*If using Per-CP-Cy5.5 labeled beads, prepare Tube B in bead dilution buffer
Tube A
Tube B
two-color setup
unlabeled
unlabeled, FITC, PE
three-color setup
using PerCP
unlabeled
unlabeled, FITC, PE,
PerCP
three-color setup
using PerCP-Cy5.5
unlabeled
unlabeled, FITC, PE,
PerCP-Cy5.5
four-color setup
unlabeled, APC
unlabeled, FITC, PE,
PerCP, APC
four-color setup
using PerCP-Cy5.5
unlabeled, APC
unlabeled, FITC, PE,
PerCP-Cy5.5, APC
6
3
FACS Loader Tube Positions
Position
Tube
1
Unlabeled beads
2
Mixed beads for LW and LNW not
containing PerCP-Cy5.5
3
Mixed beads for LW PerCP-Cy5.5 and
LNW PerCP-Cy5.5
4
HLA-B27calibration beads
5-38
Optimization samples
39
10% bleach solution (3 mL)
40
DI water (3 mL)
7
PMT Adjustments
Adjusts voltages to place a bead population
in a specific target channel
• Unlabeled beads – SSC, FL1, FL2, and
FL3 PMT adjustment
• APC beads – FL4 adjustment
8
4
FL2
Target
Channel
SSC
PMT Adjustments
Target
Channel
Target
Channel
FSC
FL1
SSC, FL1, FL2, and FL3 PMTs are set with
unlabeled CaliBRITE beads.
9
Counts
PMT Adjustments
FL4
FL4 PMT is set with APC CaliBRITE beads.
10
5
Time-Delay Calibration
blue-excited signal
blue laser (488nm)
red laser ( 635nm)
Time Delay
red-excited signal
time
11
Compensation
FL1
(530/30)
FL2
(585/42)
FL4
FL3
(661/16) (670+)
FITC
PE
PerCP
APC
12
6
FL 2
FL 2
Fluorescence Compensation
Target Mean
Difference
FL 1
FL 3
Target Mean
Difference
Target Mean
Difference
13
FL4
Fluorescence Compensation
Target
Channel
FL3
Target
Channel
FL3–%FL4
FL4–%FL3
14
7
Sensitivity
Evaluates labeled-to-unlabeled signal
separation for FL1, FL2, and FL3 parameters
FL2
FL1
Unlabeled
FITC
Unlabeled
FL3
PE
Unlabeled
PerCP
or PerCP-Cy5.5
15
Sensitivity
Evaluates PerCP-to-APC or PerCP-Cy5.5-toAPC signal separation for the FL4 parameter
FL4
PerCP or
PerCP-Cy5.5
APC
16
8
Sensitivity
Evaluates signal-to-noise separation for FSC
and SSC parameters
FSC
Noise
Singlet beads
SSC
Noise
Singlet beads
17
Assay Options
• Lyse/Wash (LW)
• LW PerCP-Cy5.5
• Lyse/No-Wash (LNW)
• LNW PerCP-Cy5.5
• HLA-B27 Calibration
18
9
Calibration Files
Assay
Choose In
Calib File Name
Set Up View
Calib File
Set Up View
Calib File.PerCP-Cy5.5
Set Up View
Calib File.LNW
Set Up View
Calib File.LNW PerCP-CY5.5
Set Up View
Calib File.B27
Preferences
CBA Calib File.LNW
Preferences
LeucoCOUNT Calib File.LNW
Location - BD Files:Instrument Settings Files
19
10
Date
Oper
UNLAB
FITC
PE
CaliBRITE
Cali-BRITELot
LotNo.
No.
BD FACSComp Log
PerCP
Signal
High
Noise
Low
FSC
P/F
P/F
SSC
FL2
FL3
Signal Separ P/F Signal Separ P/F Signal Separ P/F
FL1
SSC
FL1
FL2
FL3
337452 Rev. A
PMT Voltage
4
BD CellQuest Pro Software
After completing this module, you will be able to:
•
Acquire and analyze data
•
Perform batch data analysis
•
Generate population statistics using regions and gates
BD CellQuest Pro Software
337452 Rev. A
75
Acquisition of flow cytometry data is a multistep process. The following figure outlines
the main steps involved. In this module, we will focus on optimization of instrument
settings, data acquisition, and data analysis.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
In the following exercises, you will:
•
Optimize instrument settings for lysed whole blood samples
•
Define conditions for data acquisition and storage
•
Acquire data
•
Analyze data
•
Create a template
A peripheral blood sample stained with three-color or four-color reagents is used to
demonstrate the acquisition process. However, the steps described in the exercise can be
applied to other sample types (for example, bone marrow, cell lines, mouse cells,
monkey cells).
Before beginning this exercise, perform the following procedures.
•
System Startup on page 20 in the BD FACSCalibur System module.
•
Quality control, as described in the BD FACSComp Software module.
Optimizing Settings
In this exercise, you will optimize instrument settings as outlined in the following
graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Set up an Experiment document.
• Optimize instrument settings for your sample type.
• Save optimized settings.
76
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Setting Up an Experiment Document
You will modify the Standard Acquisition Experiment document and use it to optimize
instrument settings for data acquisition of a lysed whole blood sample. The Standard
Acquisition document is an Experiment document containing four plots and statistics
views. The document is a stationery pad. When you open a document that is a
stationery pad, a copy of the original document opens. This prevents the original from
being altered. See Locking and Using Documents as Templates on page 42 for more
information about stationery pads.
1 Click the BD CellQuest Pro software icon in the Dock to launch it.
BD CellQuest Pro icon
2 Choose File > Open Document.
3 Navigate to the Standard Acquisition document and double-click to open it.
zoom (green)
Standard Acq
document
4 Expand the Experiment window to full size by clicking the zoom button (green).
5 Choose Acquire > Connect to Cytometer (a-B).
The Acquisition Control dialog and the Acquisition view of the Browser appear.
See Figure 4-1 on page 78.
BD CellQuest Pro Software
337452 Rev. A
77
Figure 4-1 Acquisition Control and Acquisition Browser windows
NOTICE When you click on the Experiment document, the Browser will move to
the back of the active window. If the Browser is obscured from view, choose
Browser:Title X from the Windows menu to bring the window back to the front
(where Title X is the name of the Experiment document).
6 Click on the red button in the upper-left corner of the Browser window to close
the window.
7 Choose Cytometer > Detectors/Amps to display the detectors and amps controls.
8 Click and drag the Detectors/Amps window to a clear area of the screen.
When you save your BD CellQuest Pro Experiment document, the location of the
instrument settings windows (Detectors/Amps, Threshold, and Compensation)
will also be saved. The next time you open the windows, they will appear in the
last location you placed them.
Optimizing Instrument Settings
Before data is collected, you need to optimize the electronics for your samples. The
settings can be optimized with cytometer controls accessed from BD CellQuest Pro
software. The optimization sequence for immunophenotyping samples you are
acquiring is as follows:
78
•
Adjust FSC and SSC detectors
•
Adjust FSC threshold setting
•
Gate on the population of interest
•
Adjust FL1, FL2, and FL3 detector settings
•
Adjust fluorescence compensation
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Recall that BD FACSComp software generated instrument settings that are saved in the
Calib File when the Lyse/Wash assays option is selected. The settings are approximate
settings for human lymphocyte cells. Since the settings are approximate, you might need
to optimize them. If you are running cells other than human lymphocytes, instrument
settings will need to be optimized for that cell type.
The following setup samples are provided for you to perform instrument settings
optimization.
•
Isotype control
•
FITC control
•
PE control
•
PerCP control
•
APC control
Adjusting the FSC and SSC Settings
You need to adjust the FSC Amp Gain and SSC voltages to appropriately display the
scatter properties of the lysed whole blood sample. Use the FSC Amp Gain and SSC
Voltage controls to place the population of interest on scale.
In this example, the isotype control tube is used to optimize FSC and SSC settings.
However, you can use any of the setup tubes to do this step since the lightscatter
properties of cells in all tubes are similar because all tubes contain the same sample
type, lysed whole blood.
TIP Click the Pause and Restart acquisition controls periodically during the
optimization process to quickly update the events displayed in the plots.
1 Place the Isotype control tube on the sample injection port (SIP).
2 Select the flow rate and place the flow cytometer in RUN mode.
TIP
Use the MED flow rate to conserve sample.
3 Confirm that the Setup checkbox is selected in the Acquisition Control dialog,
and then click Acquire.
When the Setup checkbox is checked, you can view events in the plots displayed
without saving data.
4 Adjust the FSC Amp Gain to place the population of interest on scale in the FSC
vs SSC plot. See Figure 4-2 and Figure 4-3 on page 80.
There are several ways to adjust instrument settings. The following graphic shows
an example of the Detectors/Amps window.
BD CellQuest Pro Software
337452 Rev. A
79
Figure 4-2 Detectors/Amps window
•
Click the up or down arrow to increase or decrease the value
by 1.
•
Hold down the Option key, and click the up or down arrow to increase or
decrease the value by 10.
•
Click and hold between the two arrows to get a slider bar and drag the slider.
5 Adjust the SSC voltage to place the population of interest on scale in the FSC vs
SSC plot.
For demonstration purposes, the following graphics provide an example of cells
pre and post optimization of FSC and SSC settings. Usually, settings provided
from the Calib File need minimal adjustments to place the lymphocytes in an
optimal location in the FSC vs SSC plot.
population of
interest
before optimization
after optimization
Figure 4-3 Optimization of FSC and SSC settings
Adjusting FSC Threshold
Threshold is used to exclude unwanted events. This optimization results in a data file
that contains mainly events of interest. The samples you are running contain debris,
which are usually small and appear in the lower end of the FSC scale. Increasing the
FSC threshold gets rid of unwanted debris. For other applications, you might need to
80
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
adjust threshold for a parameter other than FSC. See examples of these other
applications in the BD MultiSET Software or DNA module.
1 Choose Cytometer > Threshold to display the Threshold controls.
2 Click and drag the Detectors/Amps window to a clear area of the screen.
3 Adjust the FSC threshold value in the Threshold window until most of the debris
is excluded.
Set the threshold to remove most of the debris without cutting off the population
of interest.
Gating on the Population of Interest
Cell populations can have different light scatter and autofluorescent characteristics. To
ensure that fluorescence instrument settings are optimized for only the population of
interest, draw a region around the lymphocytes and view only the gated events when
optimizing fluorescence PMT settings.
1 Click the Polygon-Region (
) tool in the tool palette to select it.
2 Create a region around the population of interest in the FSC vs SSC plot. See
Figure 4-4 on page 82.
•
Move the cursor over the plot on which you want to create the region. Note
that the cursor changes to a crosshair. Click to set the first vertex.
•
Release the mouse button and move the cursor to where you want the second
vertex and click again.
•
Continue this way until the population of interest is encompassed by the
region. You can close the region either by double-clicking or by clicking on
the first vertex.
Notice that clicking in a plot makes it active (colored border). When a plot is
active, you can create regions and display different data, if needed. You cannot
delete, move, or resize an active plot.
BD CellQuest Pro Software
337452 Rev. A
81
3 Click outside the region to deselect it, then click and drag the region label R1 to a
new location on the plot away from the data.
This prevents data from being obscured by the label.
Figure 4-4 R1 around population of interest
4 Change the color of the gated population, if needed.
Red dots on a black background might be difficult to view. If needed, you can
change the color of the gated population by doing the following.
•
Choose Gates > Gate List.
•
Click in the color box next to the G1 label.
•
Select a different color in the dialog that appears.
•
Close the Gate List.
5 Remove the Isotype control tube.
The tube is removed to conserve sample while you prepare to do the next
optimization step.
Adjusting the Fluorescence Detector Settings
The FL1, FL2, and FL3 detector settings might need adjustment to place the negative
population in the lower-left corner of the plot. Placing the negative population in the
lower left leaves plenty of room for the positives on the x- and y-axes.
Mouse anti–keyhole limpet hemacyanin (KLH) antibodies are used in the isotype
control. When these antibodies are added to human cells, any binding that occurs is
non-antigen specific.
82
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Antibody binding which is not antigen specific can occur in two ways. The first is when
the Fc portion of the antibodies bind to the Fc receptors on the cell membrane. The
second is when the antibody sticks to the cell due to physical interactions, independent
of the antigen specificity of the antibody. This in combination with autofluorescence is
considered background fluorescence. Specific binding of antibodies occurs when the
Fab portions of the antibodies bind to antigens on the cell surface to which particular
antibodies are directed. This is considered antigen-specific binding.
1 Select all the plots except the FSC vs SSC plot.
To select a plot, click on the border of the plot. Square handles appear.
selection handles
To select multiple plots, hold down the Shift key while clicking on the border of
each plot.
When a plot is selected, you can click on its border and move it. You can also
click on a square handle and resize it. Delete by pressing Delete or choosing Edit >
Cut or Clear.
2 Choose Windows > Show Inspector.
3 Choose G1 = R1 from the Gate pop-up menu in the Inspector.
Plots display only events that are in gate G1.
BD CellQuest Pro Software
337452 Rev. A
83
NOTICE Contents in an Inspector change, depending on the object(s) selected. An
Inspector only displays information when one or more objects is selected, not
active.
4 Verify that the mode for FL1, FL2, FL3 and FL4 parameters are set to LOG in the
Detectors/Amps window.
5 Click the quadrant marker intersection in the FL1 vs FL2 plot to select it.
These markers will designate areas of negativity and positivity.
6 Drag the handle of the markers so the cross-hairs are at 101 and 101 on the x- and
y-axes, respectively.
Changing Parameters Displayed on Plots
To practice changing parameters displayed on a plot, do the following.
1 Reverse the X and Y parameters on the FL2 vs FL3 plot to FL3 vs FL2.
2 Change the FL1 vs FL3 plot to FL3 vs FL4.
Adjusting Settings
1 Create quadrant markers and display quadrant statistics for the FL3 vs FL2 and
FL3 vs FL4 plots.
2 Install the Isotype control tube on the SIP.
3 Click Pause, Restart in the Acquisition Controls dialog.
4 If necessary, adjust the FL1, FL2, FL3, and FL4 voltages in the Detectors/Amps
window to place the negative population in the lower-left quadrant.
5 Click Pause in the Acquisition Control dialog.
6 Remove the Isotype control tube from the SIP.
7 Click the close boxes of the Detectors/Amps and Threshold windows to remove
the windows from the screen.
84
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Adjusting Fluorescence Compensation
The last step in the optimization process is adjusting compensation to correct for
spectral overlap.
It is important to choose appropriate reagents for compensation. Selection should be
based on the panel of reagents you are using. Compensation for each color should be
set using the brightest stained population. For example, in a panel containing
CD3 FITC, CD4 FITC, and CD8 FITC, the FITC compensation should be set using
CD8 FITC, the brightest FITC-stained population in the panel.
Compensation adjustments are dependent on the detector voltages established on the
isotype control. Once the voltages are set, compensation is adjusted on the fluorescencepositive cells.
1 Choose Cytometer > Compensation to display compensation controls.
2 Click and drag the Compensation dialog to a clear area of the screen, if needed.
3 Place the FITC control tube on the SIP.
4 Click Restart in the Acquisition Control dialog.
5 If necessary, adjust FL2–%FL1 to place the FITC+ population in the lower-right
quadrant.
Align the FITC+ population with the negative population.
before optimization
after optimization
6 Remove the tube from the SIP and place the PE control tube on the SIP.
7 If necessary, adjust FL1–%FL2 to place the PE+ population in the upper-left
quadrant. See Figure 4-5 on page 86.
Align the PE+ population with the negative population.
BD CellQuest Pro Software
337452 Rev. A
85
before optimization
after optimization
Figure 4-5 PE compensation
8 If necessary, adjust FL3–%FL2 to place the PE+ population in the upper-left
quadrant.
Align the PE+ population with the negative population.
before optimization
after optimization
9 Remove the tube and place the PerCP control tube on the SIP.
10 If necessary, adjust FL2–%FL3 to place the PerCP+ population in the lower-right
quadrant.
It should be 0.0% because PerCP usually does not emit within a range that
overlaps into the FL2 detector.
86
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
11 If necessary, adjust FL4–%FL3 to place the PerCP+ population in the lower-right
quadrant.
before optimization
after optimization
12 Remove the tube and install the APC control tube on the SIP if you are running
four-color samples. If you are running three-color samples, proceed to step 14.
13 If necessary, adjust FL3–%FL4 to place the APC+ population to the left of the
vertical marker.
Align the APC+ population with the negative population.
before optimization
after optimization
14 Click the close box of the Compensation dialog to remove it from the screen.
15 Click Pause, then Abort in the Acquisition Control window.
16 Remove the tube from the SIP.
17 Place a tube of DI water on the SIP.
18 Place the cytometer in standby mode.
BD CellQuest Pro Software
337452 Rev. A
87
Saving an Experiment Document
In BD CellQuest Pro software, the Experiment document you have set up with plots,
gates, markers, and statistics can be saved. The document can be opened later to resume
acquisition or analysis.
1 Choose File > Save Document.
2 Select the appropriate folder.
To select a destination, you can click the Where drop-down menu and make a
selection.
Alternatively, you can click the scroll bar to navigate to a destination choice. If
the scrollbar is not visible, click the display/hide button.
display/hide button
scroll bar arrows
88
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
3 Enter a name for the file.
For example, enter Optimization Document as the name for the document.
4 Click Save.
Saving Instrument Settings
The optimized instrument settings can be saved and later restored to the flow cytometer
when you run lysed whole blood samples again.
1 Choose Cytometer > Instrument Settings.
If the Instrument Settings menu item is grayed out, click Pause and then Abort in
the Acquisition Control dialog.
2 Click Save in the Instrument Settings window. See Figure 4-6 on page 89.
Figure 4-6 Instrument Settings dialog
3 Select the appropriate folder.
4 Enter a name for the settings file or use the default name. See Figure 4-7 on
page 90.
BD CellQuest Pro Software
337452 Rev. A
89
Figure 4-7 Save instrument settings dialog
5 Click Save.
6 Click Done in the Instrument Settings window.
Restoring Instrument Settings
You can retrieve instruments settings in one of two ways.
•
From an instrument settings file.
•
From an FCS data file.
Practice retrieving instrument settings from the file you just saved by doing the
following.
1 Choose Cytometer > Instrument Settings
2 Click Open.
3 Locate the instrument settings file or FCS file in the location dialog that appears.
4 Select the appropriate file, then click Open.
The new settings appear in the Instrument Settings window.
5 Click Set in the Instrument Settings window to send these settings to the
cytometer.
NOTICE You must click Set to download instrument settings; otherwise, previous
settings will still apply.
Previous settings can be returned to the cytometer by clicking Revert.
6 Click Done.
90
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Acquiring Data
In this exercise, you will acquire data as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Specify data collection information (file name, storage location, parameter labels, tube-specific settings,
and so on).
• Display counters, if needed.
• Deselect Setup checkbox.
• Click Acquire.
Specifying Data Collection Information
In this section, you will do the following.
•
Specify data file name and storage location.
•
Specify acquisition and storage settings
•
Create and use a panel (optional).
•
Specify tube-specific settings (optional).
Before you save data, you need to specify information such as data file name, storage
location, parameter labels, number of events to save, and so on.
Specifying Data File Name and Storage Location
File storage and parameter labels are defined in the Acquisition view of the Browser.
1 Make sure your Experiment document is open.
If needed, double-click your Experiment document’s icon to open it or choose
File > Open Document and then navigate to the location where your document
was saved and double-click to open it.
2 Choose Windows > Show Browser. See Figure 4-8 on page 92.
Click the disclosure triangle next to Acquisition Controls in the Browser window
to view the Acquisition Controls, if needed.
BD CellQuest Pro Software
337452 Rev. A
91
File (name) Change
button
Directory Change button
disclosure triangle
Figure 4-8 Acquisition Browser
3 Click the Directory Change button.
4 Select your folder.
5 Click Choose.
6 Click the File Change button.
7 Enter a file name in the highlighted Custom Prefix field in the File Name Editor
dialog that appears.
8 Verify File Count is selected from the File Name Suffix pop-up menu; click OK.
9 Enter Donor 1 in the Sample ID field in the Acquisition Browser.
10 Choose File > Save Document As.
11 Enter a name for the document, choose your folder, and then click Save.
92
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Specifying Acquisition and Storage Settings
Use Global Acquisition & Storage settings to define the number and type of events to
acquire and store. Global acquisition settings apply to all tubes in an Experiment
document that do not have specific settings associated with them. For tube-specific
settings, see Specifying Tube-Specific Settings (Optional) on page 99.
1 Display acquisition and storage settings.
There are two ways to display acquisition and storage settings.
•
In the Browser, double-click the Global Acquisition & Storage Settings icon.
The Acquisition & Storage Inspector window displays the current settings.
Acquisition
Browser
icon
Acquisition
& Storage
Inspector
•
Choose Acquire > Acquisition & Storage. The Acquisition & Storage dialog
appears.
BD CellQuest Pro Software
337452 Rev. A
93
2 Make selections in the Acquisition & Storage dialog or in the Inspector.
For this example, make the following selections.
•
Leave the defaults Accept and All in the Acquisition Gate pop-up menus.
•
Enter 2,000 and choose G1 = R1 in the Collection Criteria.
•
Leave the default All as the Storage Gate.
•
Leave the Resolution 1024.
Based on the above selections, data collection will stop when a count of 2,000 is
reached within gate G1, but all data is saved. Refer to the BD CellQuest Pro
Software User’s Guide for additional information about settings in the
Acquisition & Storage window or Inspector.
3 Click OK to exit the Acquisition & Storage window, if needed.
4 Save the Experiment document.
5 Continue with one or more of the following sections or proceed to Saving Data
on page 103.
•
Specifying Parameter Labels (Optional) on page 94
•
Specifying Tube-Specific Settings (Optional) on page 99
Specifying Parameter Labels (Optional)
Parameter labels allow you to identify parameters with specific names instead of FL1,
FL2, FL3, FL4, and so on. This is useful when you analyze data because you know
exactly what was in the tube you acquired.
There are two methods you can use to specify parameter labels.
•
Method 1–Enter the labels before each tube is placed on the cytometer.
•
Method 2–Create a pre-defined list of labels and save it as a reagent panel that
can be re-used. See Method 2–Creating a Reagent Panel on page 95 for more
details.
Use method 1 if you are acquiring tubes stained with unique reagent combinations for
every experiment or donor. Use method 2 if you are acquiring the same reagent
combination for every experiment or donor.
Follow the steps for method 1 or method 2, not both.
94
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Method 1–Entering Labels Before Each Tube Is Acquired
1 Enter a label for P1 or choose a pre-defined label from the pop-up menu.
In the box next to P1, type a description, for example, FSC. Alternatively, click on
the pop-up menu and select a P1 label, such as Forward Scatter.
2 Define labels for the other parameters.
For example, enter the following.
•
P2: Side Scatter
•
P3: FITC
•
P4: PE
•
P5: PerCP
•
P7: APC (if you are running four-color samples)
NOTICE Labels can be added or deleted from each parameter label pop-up menu
by choosing Acquire > Edit Reagent List. Refer to the BD CellQuest Pro Software
User’s Guide for instructions.
Method 2–Creating a Reagent Panel
A panel is a list of tubes with predefined parameter labels. You will define one of the
following three-tube panels.
Three-Color Panel
•
FITC Control/PE Control/CD45 PerCP (PerCP Control)
•
CD3 FITC/CD8 PE/CD45 PerCP
•
CD3 FITC/CD16+CD56 PE/CD45 PerCP
BD CellQuest Pro Software
337452 Rev. A
95
Four-Color Panel
•
FITC control/PE control/CD45 PerCP/APC control (PerCP Control)
•
CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC
•
CD3 FITC/CD16+CD56 PE/CD45 PerCP/CD19 APC
1 Choose Acquire > Edit Panels.
Add panel
button
2 Click Add above the Panels column.
A new panel appears under the existing panel.
3 Enter a name for the panel.
4 Click the selection icon to the left of the panel name to select the panel and click
Add above the Tubes column. See Figure 4-9 on page 97.
Tube #1 is added.
96
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Add tube button
label pop-up
menu
selection icon
Figure 4-9 Edit Panel dialog
5 Change tube 1’s name to CD45control.
6 Click the selection icon to the left of the CD45 control.
7 Choose Forward Scatter as the label for the P1 and Side Scatter as the label for P2
from the label pop-up menu under the Labels column.
8 Enter fluorescence parameter labels for tube 1.
For example, enter the following.
•
P3: FITC control
•
P4: PE control
•
P5: CD45 PerCP
•
P7: APC control (if you are running four-color samples)
9 Click Add above the Tubes column.
10 Enter 3/8/45/4 as the tube name and choose parameter labels for this tube.
For example, enter the following.
•
P1: Forward Scatter
•
P2: Side Scatter
•
P3: CD3 FITC
BD CellQuest Pro Software
337452 Rev. A
97
•
P4: CD8 PE
•
P5: CD45PerCP
•
P7: CD4 APC (if you are running four-color samples)
11 Click Add above the Tubes column.
12 Enter 3/16+56/45/19 and choose parameter labels for this tube.
For example, enter the following.
•
P1: Forward Scatter
•
P2: Side Scatter
•
P3: CD3 FITC
•
P4: CD16+CD56 PE
•
P5: CD45 PerCP
•
P7: CD19 APC (if you are running four-color samples)
13 Click OK to exit the Edit Panel dialog.
14 Click the pop-up control next to Untitled Acquisition Tube List and choose Load
Tubes from Panel; select the panel you just created.
If the Browser is not already opened, choose Windows > Browser: untitled.
Note that your browser might appear different from the one shown here,
depending on whether or not the Acquisition Controls portion of the Browser is
open or closed.
15 Click the disclosure triangle next to the panel folder icon to display the tubes in
the panel.
98
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
A list of tubes appears. A pointer points to the first tube in the panel.
16 Save the Experiment document.
17 Continue with Specifying Tube-Specific Settings (Optional) or proceed to Saving
Data on page 103.
Specifying Tube-Specific Settings (Optional)
BD CellQuest Pro software allows you to save settings specific for a tube. For example,
each tube can have the following specific settings.
•
Acquisition and storage settings
•
Instrument settings
•
Associating plots with tubes (only available with panels)
Specifying Tube-Specific Acquisition & Storage Settings
You might want specific settings, such as the number of events to acquire, for a
particular tube. To do this, do the following.
1 Click once on the pop-up control next to a tube in the tube list.
2 Choose Add Acquisition & Storage Settings from the menu that appears.
BD CellQuest Pro Software
337452 Rev. A
99
An icon for Acquisition & Storage Settings appears indented, beneath the tube.
Click once on the disclosure triangle next to the tube name to display the icon, if
needed.
Global Acquisition & Storage icon
Acquisition & Storage icon
Note that the icon is the same as the one listed beneath Global Settings; however
the name next to the icon is Acquisition & Storage Settings rather than Global
Acquisition & Storage Settings. These settings are now specific only to the second
tube.
3 Double-click the Acquisition & Storage icon under a tube in the Browser to
display the Acquisition & Storage Inspector.
The Inspector displays the current acquisition and storage settings.
4 Make any necessary changes to the acquisition and storage settings.
All changes apply as they are entered.
5 Repeat steps 1 through 4 for any other tube-specific settings.
6 Continue specifying other tube-specific settings or proceed with Saving Data on
page 103.
Specifying Tube-Specific Instrument Settings
You might want to specify different instrument settings for certain tubes in a panel. For
example, if you are running samples stained with PerCP and PerCP-Cy5.5 reagents, you
might want to associate different instrument settings with tubes containing PerCP
reagent than those containing PerCP-Cy5.5 reagent.
1 Click once on the pop-up control next to a tube in the tube list.
100
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
2 Choose Add Instrument Settings from the pop-up control next to a tube in the
Browser.
An Instrument Settings icon is added below the tube name. The icon is crossed
out until instrument settings are defined. Click once on the disclosing triangle
next to the tube name to display the icon, if needed.
missing instrument
settings icon
3 Double-click the new Instrument Settings icon to display the Instrument Settings
Inspector.
4 Click the File button and select the appropriate instrument settings file in the
location dialog box that appears.
The file name appears in the Inspector.
5 Repeat steps 1 through 4 for any other tube-specific settings.
6 Continue specifying other tube-specific settings or proceed with Saving Data on
page 103.
Creating Plots and Associating Them With Tubes
After defining an acquisition tube list, you will need to create appropriate plots on your
Experiment document. You can then associate the plots with specific tubes. During
acquisition, data will be displayed in the associated plots and remain on display until all
tubes in the panel are acquired.
BD CellQuest Pro Software
337452 Rev. A
101
1 Verify that a panel has been loaded in the Acquisition view of the Browser.
2 Verify that all plots are Acquisition -> Analysis plots.
•
Select all plots.
•
Select Acq -> Analysis from the Plot Type pop-up menu in the Inspector.
3 Select the FSC vs SSC and the FL1 vs FL2 plots in the Experiment document or in
the Acquisition Browser.
You can select the plot(s) in the Experiment document or select one or more plot
icons in the Browser. Hold down the Shift key to select multiple plots that are not
listed next to each other in the plot list.
4 Click the Tube pop-up menu in the Inspector and choose tube #1.
After the plot(s) have been associated with a tube, the tube list in the Browser
displays the plots under the tube icon.
plots associated
with tube
5 Continue creating plot(s) and associating tubes with plot(s) until each tube in the
panel has been associated.
102
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Saving Data
At this point, the electronics have been optimized for your cells, the gate and number of
events to acquire and store have been established, and file storage has been defined.
Data for each tube can now be saved.
1 Choose Acquire > Counters to display the Counters window, if needed.
The Counters window has two sizes. Use the enlarged window if you are
acquiring gated data.
zoom button
2 Move the window to a clear area of your screen.
3 Deselect the Setup checkbox in the Acquisition Control window.
4 Verify the following.
•
Tubes in the Tubes List are in the order you want.
If needed, you can alter the order of the tubes listed and add or delete tubes.
Refer to the BD CellQuest Pro Software User’s Guide for details.
•
Acquisition pointer is pointing to the appropriate tube in the Browser.
•
Parameter labels are correct in the upper pane of the Browser.
5 Place the flow cytometer in RUN mode.
6 Install the first tube on the SIP; immediately place the support arm under the
sample tube.
BD CellQuest Pro Software
337452 Rev. A
103
7 Wait approximately 5 seconds before clicking Acquire.
There is a brief period during which the sample tube is being pressurized. It is
important to wait for the pressure in the sample tube to stabilize before clicking
Acquire.
8 Click Acquire.
Live quadrant statistics will be displayed as the data is acquired. The system beeps
at the completion of acquisition.
9 Remove the tube after the beep.
10 Verify the parameter labels are correct and place the next tube on the SIP.
11 Wait approximately 5 seconds and click Acquire; remove the tube after the beep.
12 Repeat steps 10 and 11 for the remaining tube in your panel.
13 Install a tube of DI water on the SIP and place the flow cytometer in standby
mode.
Analyzing Data
In this exercise, you will perform data analysis as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Display data.
• Create gates and statistics, if needed.
• Create user-defined mathematical expressions, if needed.
• Perform data batch analysis, if needed.
• Review the results.
• Print or export results, if needed.
Adding a second, third, or fourth color in each tube increases the amount of
information that can be obtained and can help reduce the number of tubes that need to
be acquired.
Although this exercise uses human blood cells to demonstrate the analysis features of
BD CellQuest Pro software, you can apply the same principles to analyze other sample
types.
104
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Displaying Data
Consult the appropriate software user’s guide if you want to analyze data using
BD Attractors™ or BD PAINT-A-GATEPRO™software.
In this exercise, you will create dot and contour plots. Other plot types, like density,
histogram, and 3-D, can also be used. Consult the BD CellQuest Pro Software User’s
Guide for information about these other plot types.
1 Launch BD CellQuest Pro software, if necessary.
If BD CellQuest Pro software is open, select File > New Document.
2 Expand the Experiment window to full size by clicking the zoom button.
Creating and Displaying Data in a Dot Plot
1 Select the Dot-Plot tool (
) from the tool palette.
The Dot-Plot tool becomes highlighted.
2 Click in a clear area of the Experiment window and drag diagonally until the plot
outline is an appropriate size.
When you let go of the mouse, a dot plot and the Dot Plot Inspector appear.
3 Choose Select File from the File pop-up menu in the Inspector.
The Open a Data File dialog box appears.
NOTICE To display FCS data files, you must first create a plot and then choose
Select File from the Inspector. You cannot view an FCS data file by doubleclicking its icon.
4 Navigate to your folder, select the first tube’s data file, and then click Open.
BD CellQuest Pro Software
337452 Rev. A
105
Only those files appropriate for the plot type are displayed in the dialog box.
Single-parameter histogram files will not appear.
5 Double-click the data file of the first tube acquired.
In the training class, the first data file contains data from a sample stained with
one of the following.
•
FITC control/PE control/CD45 PerCP
•
FITC control/PE control/CD45 PerCP/APC control
6 Choose CD45 PerCP as the X parameter and SSC-H as the Y parameter.
7 Click the MultiColor Gating checkbox under Dot Plot in the Inspector.
Multicolor Gating checkbox
Creating and Displaying Data in Contour Plots
1 Choose Plots > Contour Plot.
A second plot appears and the Inspector changes to a Contour Plot Inspector.
2 Choose Select File from the File pop-up menu in the Inspector.
3 Navigate to your data file folder and open the data file from the first tube
acquired.
4 Choose FITC control as the X parameter and PE control as the Y parameter.
5 Click the MultiColor checkbox next to Color in the Inspector.
If necessary, drag the plot to a clear area of the document.
Multicolor Gating checkbox
106
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Creating Gates and Displaying Statistics
Next, you will draw a gate around the population of interest. You will also create
quadrants to distinguish areas of negativity and positivity and display quadrant
statistics.
The Snap-To–Region tool will be used to create a region. Other region types can also be
used, like polygon, rectangle, ellipse, and histogram. Refer to the BD CellQuest Pro
Software User’s Guide for more information about regions.
NOTICE Snap-To regions cannot be created in Acquisition only plots. You cannot use
a Snap-To region as an acquisition gate.
Creating a Snap-To Region
1 Draw a region around the population of interest in the CD45 vs SSC plot using
the Snap-To–Region tool (
) in the tool palette.
To set the region, click the tool in the tool palette, and then click on the
population. The region is automatically drawn. See Figure 4-10.
NOTICE If the cluster is too small or diffuse for the Snap-To–Region tool to
recognize, the system beeps and no region is drawn. In this case, use another
region tool to manually draw the region.
To delete a region, select the region and choose Edit > Cut or press Delete.
Figure 4-10 Snap-To region around population of interest
2 Click anywhere outside of the region to deselect it, then drag the R1 symbol off to
the side of the region.
3 Hide the region outline, if needed.
BD CellQuest Pro Software
337452 Rev. A
107
To hide the region outline, click anywhere in the plot while pressing the
Command key (a). A pop-up menu appears. Choose the specific region to hide if
there is more than one region or choose Hide All Regions.
Creating Quadrant Markers
Quadrant markers will be set around the gated data in the contour plot.
1 Select the contour plot and choose G1=R1 from the Gate pop-up menu in the
Inspector.
The plot displays only data from the G1=R1 gate.
2 Draw quadrant markers on the FITC control vs PE control plot.
Select the quadrant-marker tool (
) in the tool palette and then click and drag
in the contour plot until the markers appear. Set the marker so the isotype
population is in the lower-left (LL) quadrant.
3 Choose Stats > Quadrant Stats.
The plot must be active for the statistics view to appear. Following is an example
of a Quadrant Statistics view. See Figure 4-11 on page 109.
108
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 4-11 Quadrant Statistics view
The contour plot is divided into four quadrants. The fluorescence of each
quadrant in the FL1 vs FL2 plot is as follows.
UL
Upper Left
FL2+ only
UR
Upper Right
FL1+ and FL2+
LL
Lower Left
FL1– and FL2–
LR
Lower Right
FL1+ only
In the contour plot for the isotype control, most of the events fall in the lower-left
quadrant. As shown in the Quadrant Statistics view, the % Gated for the LL
quadrant is 99.68%. This means that 99.68% of the events are negative for FL1
and FL2. This is expected because the isotype control accounts for nonspecific
binding of antibodies.
4 Edit the type of statistics displayed.
•
Select the statistics view.
•
Choose Stats > Edit Quadrants Stats.
•
Make selections in the Edit Quadrant Statistics dialog.
Click in the box to the left of the statistic to deselect or select that statistic.
Since the statistics view usually contains more information than you will need
for spreadsheet analysis, deselect some of the extraneous information.
Additionally, you can display header information in one or two columns.
•
Click OK to accept the changes and to exit the dialog.
BD CellQuest Pro Software
337452 Rev. A
109
Statistics that are checked will appear in the statistics view. Only the statistics you
choose in the Edit Stats dialog box are exported.
5 Reduce the size of the statistics view, if needed.
•
Select the statistic view, if it is not already selected.
•
Click on one of the square handles, and then drag the handle to decrease the
size of the view.
6 Click the frame of the Quadrant Statistics view and drag it to a clear area of the
document, if necessary.
7 Look at the Analysis view of the browser.
If the view is hidden behind the experiment window, choose Windows >
Browser:untitled.
Click the disclosure triangle next to the folder icon and the tube icon to view all
the items in the Analysis browser.
Notice that the view now displays the isotype control tube, and the plots
currently displaying the data file from the tube are listed under the tube’s icon.
110
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
; Tip To quickly navigate to a particular plot within an Experiment document
containing several pages, Option-Click on a plot icon in the Analysis Browser.
If you are analyzing three-color data files, proceed to one of the following sections.
•
Using the Hilite Feature (Optional) on page 111.
•
Using the Expression Editor (Optional) on page 113.
•
Batch Analysis of the Remaining Data Files on page 115.
If you are analyzing four-color data files, continue with the following steps.
Creating Additional Plots
1 Click on the Experiment document to make it the active window.
2 Choose File > Document Size.
The document Size dialog box appears.
3 Click the rectangle to the right of the current selection, then click OK.
The view area increases to a two-page width.
4 Select the FITC control vs PE control contour plot and choose Edit > Duplicate.
A duplicate plot appears.
5 On the contour plot you just created, change the parameters to FITC control vs
APC control.
6 Set the quadrant marker so the isotype population is in the lower-left (LL)
quadrant in the FITC control vs APC control plot.
7 Display quadrant statistics for the FITC control vs APC control plot.
Choose Stats > Quadrant Stats. If necessary, move the stats box to a clear area of
the screen.
Using the Hilite Feature (Optional)
When the Hilite box is checked in the Gate List window, any multicolored events in the
selected gate are drawn larger throughout the Experiment document, making them
more prominent and easier to view. Use the Hilite feature to locate a rare population on
a dot plot or to differentiate between a mixed population within a region. You can
highlight up to sixteen populations. With this feature, you can create plots highlighting
populations of interest for analysis and publication purposes.
BD CellQuest Pro Software
337452 Rev. A
111
NOTICE
Highlighting does not add events nor does it affect statistics.
; TIP
You can export plots in a variety of graphical formats and save Experiment
documents as PDF files, if needed. Adobe® Acrobat® PDF Writer application is needed
to create PDF files. Refer to the BD CellQuest Pro Software User’s Guide for details.
To practice using the Hilite feature, do the following:
1 Create a region around the monocyte population (R2).
Notice that region R1 border is thicker than region R2 label. This indicates that
region R1 is a Snap-To region.
2 Choose Gates > Gate List.
The Gate List appears.
3 Click the G2 checkbox in the Gate List to select the Hilite option.
4 View the dot plot to see the change.
5 Delete region R2.
Select the region, then press Delete.
112
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Using the Expression Editor (Optional)
In this exercise, you will use the Expression Editor to create mathematical expression to
calculate total CD3+ T cells. Expressions update automatically as data in the document
changes.
1 Verify that quadrant labels and % Gated data are displayed in the statistics view.
quadrant labels
2 Select the Expression Editor tool (
) in the tool palette.
The Expression Editor dialog box appears.
expression box
3 Enter %Total CD3+ cells = in the Label field.
BD CellQuest Pro Software
337452 Rev. A
113
To prevent text from running together with the calculated value, enter a space at
the end of the text entered in the Label field.
4 Click to place the cursor inside the expression box.
5 Click the UR % Gated value in the statistics view.
The value appears in the expression box.
6 Click the plus sign (
) in the Expression Editor.
The + sign appears next to the UR % Gated value in the expression box.
7 Click the LR % Gated value.
The value appears in the expression box. Note that the calculated result appears
in the dialog box. See Figure 4-12 on page 115.
114
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
calculated result
Figure 4-12 Expression Editor dialog
8 Click OK.
The result appears in the Experiment document. Note the double lines
surrounding the text and the square handles. The square handles indicate that the
text is selected. You can resize and move a selected text box. Use the Inspector to
make changes to font type, size, and color.
NOTICE The double lines appear on the screen but are not printed.
The first data file is the isotype control, so the % Total CD3+ T-cell value is
meaningless for this file.
Batch Analysis of the Remaining Data Files
In this part of the exercise, you will use the batch analysis feature in BD CellQuest Pro
software to analyze the remaining data files.
Once you set up the Experiment document with plots, gates, markers, and statistics,
BD CellQuest Pro software can apply the same analysis to subsequent files by batch. In
addition to advancing data files for the plots, batch analysis can automatically pause
after each execution for you to adjust markers or regions while the batch is paused.
Batch analysis can also provide automatic printing of the document and automatic
export of statistics to an export file. To view an example of opening an export file in a
BD CellQuest Pro Software
337452 Rev. A
115
third-party spreadsheet application, see Importing Statistics into a Spreadsheet on
page 119.
The software also provides the Load Sample feature, which is useful for managing the
analysis of large, multi-tube data files, especially when the files were acquired as part of
a panel. This feature is not shown here. For details, refer to the BD CellQuest Pro
Software User’s Guide.
1 Choose Batch > Setup.
2 Make the following selections in the dialog that appears.
•
When the All radio button is selected under Plots and Stats to Process field,
data will be processed through all plots in the document.
•
When the for [n] seconds radio button is selected under Pause after each file
increment field, the batch will pause for n seconds after each data file is
processed.
•
When the Print after each file increment checkbox is checked, the Print One
command is executed after each data file is processed.
•
When the Export statistics checkbox is checked, all statistics (eg, quadrant
and histogram statistics) from the document are exported to a file that you
can open in a third-party application such as Microsoft Excel.
•
The number in the File Increment field instructs the software to increment
that number of data files when batching. For example, if 2 is entered in the
field and the first data file is .001, the next data file analyzed would be .003.
3 Click the New File button.
The save dialog box appears.
116
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 Enter a name for the export file in the Save as: field.
For this exercise, enter Stats 1 as the file name.
5 Choose the folder destination, then click Save.
In the training class, choose your folder in the Student folder.
6 Click OK.
7 Choose Batch > Run.
A Batch Control floater appears. Click Stop, Pause, or Resume anytime during
the run if you want to interrupt batch analysis.
stop
pause
resume
A message box appears at the end of the batch run to notify you when the batch
run is finished.
Reviewing Analysis Results
You will use your printouts to calculate the percentage of lymphocytes, CD3+ cells,
CD19+ cells, and CD16+CD56+ cells.
1 From the first data file, report the % Gated events in the Lower Left (LL)
quadrant of the FITC control vs PE control plot.
% LL ______________
2 If you acquired four-color data, report the % Gated events in the Lower Left (LL)
quadrant of the FITC control vs APC control plot from the first data file.
% LL ______________
BD CellQuest Pro Software
337452 Rev. A
117
3 From the second and third data files, report the percentage of lymphocytes that
are CD3+ (% Gated CD3+ cells).
% CD3+ cells______________ (second data file)
% CD3+ cells______________ (third data file)
Are the percentages within 5% of each other? __________ (consistency check)
4 From the second data file, calculate the percentage of lymphocytes that are
CD3+CD8+ cells.
% CD3+CD8+ cells______________
5 If you acquired four-color data, calculate the % CD3+CD4+ cells from the second
data file.
% CD3+CD4+ cells______________
6 From the third data file, calculate the percentage of lymphocytes that are CD3–,
CD16+CD56+ cells.
% CD3–, CD16+CD56+ NK cells______________
7 If you acquired four-color data, calculate the % CD3–, CD19+ cells from the third
data file.
% CD3–, CD19+ cells______________
Saving an Experiment Document as a Template
A BD CellQuest Pro software document can be saved as a generic template so it can be
used for routine acquisition or analysis work. All plots, regions, markers, statistics, and
so on (except data files) can be defined and saved as a template.
1 Select all plots in the Experiment document.
To select multiple plots, press the Shift key and click on the border of each plot.
Square handles appear on all selected plots.
2 Click the File pop-up control in the Inspector and choose No File from the popup menu.
118
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Each plot is now empty. The next time you open the Experiment document, the
plots remain empty until data are read in.
3 Choose File > Save Document As.
4 Enter a file name for your new Experiment document.
5 Make sure an appropriate destination folder is displayed in the dialog box and
click Save.
6 Lock the document or make it a stationery pad document, if needed.
•
Select the Experiment icon.
•
Choose File > Get Info.
•
Select the Stationery Pad or Locked checkbox.
See Locking and Using Documents as Templates on page 42 for additional
information about stationery pad and locked documents.
Importing Statistics into a Spreadsheet
In this exercise, you will import the export file you saved during your data analysis into
a third-party spreadsheet application.
1 Choose Microsoft Excel from the Dock.
A blank worksheet appears on the screen.
2 Choose File > Open.
A dialog box appears.
3 Navigate to the location of your export file and open it.
The file opens and displays the statistics in a spreadsheet format.
4 Click Finish on the Text Import Wizard window.
5 View the results.
6 When you are finished viewing the statistics, choose File > Quit.
BD CellQuest Pro Software
337452 Rev. A
119
Using Regions and Gates
A region is a boundary drawn around a subpopulation to isolate events. BD CellQuest
Pro software allows you to set five types of regions: rectangular, elliptical, polygonal,
histogram, and Snap-To regions.
In the following exercises, you will practice using region and gate features in
BD CellQuest Pro software by doing the following.
•
Creating and altering regions
•
Using Snap-To regions
•
Using region statistics to analyze data
•
Using gates
•
Using combined gates
•
Using combined gates to analyze histograms
Additional information about regions and gates can be found in the BD CellQuest Pro
Software User’s Guide.
Creating Regions
To create a region, click on the appropriate tool in the tool palette, then draw the region
on the plot of interest.
1 Navigate to the Gating Exercise document in the Gating folder in the Training
Files folder on your hard drive, and double-click it.
120
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Since you are opening a stationery pad document, a copy of the original
document will open.
2 Draw a polygonal region (R1) around the lymphocyte cluster on the FSC vs SSC
dot plot.
granulocytes
monocytes
lymphocytes
3 Drag the R1 symbol off to the side of the region.
This prevents it from being obscured by data.
Altering a Region
You can alter a region by resizing, repositioning, or rotating it. For polygonal regions,
you can reposition individual vertices. You can also display or hide a region’s boundary.
You will practice altering regions in the following exercises.
Resizing a Region
1 Click once on the boundary of the region you just created.
Four handles will appear, one at each corner of the region. You can click and drag
these handles to stretch the region along the x- and y-axes.
handles
Handles
2 Click and drag the upper right handle to stretch the region so that it encompasses
the monocyte cluster. See Figure 4-13 on page 122.
BD CellQuest Pro Software
337452 Rev. A
121
Figure 4-13 R1 resized
3 Return the region to its original position by clicking and dragging on the upper
right handle.
Repositioning a Region
While the region is selected, you can also click and drag anywhere on the border of the
region to reposition it. Try this with region 1.
1 Select region R1 if it is not already selected.
2 Click and drag on the region border to position it over the monocyte cluster.
NOTICE Do not drag the handles to reposition a region. Drag the region border.
3 Return region R1 to its position around the lymphocytes.
Rotating a Region
Rotate a selected region by first choosing Rotate Region from the Gates menu, and then
clicking and dragging on a handle to rotate the region. When you have finished rotating
the region, select Stop Rotating Region from the Gates menu, or click anywhere in the
plot outside the region.
122
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
1 Select region R1 if it is not already selected.
2 Choose Gates > Rotate Region.
3 Drag one of the region’s handles to rotate the region 90° counterclockwise.
4 Reposition region R1 in its original orientation.
You can also return the region to its original position by selecting Undo from the
Edit menu.
5 Select Gates > Stop Rotating Region.
You can also rotate a region without selecting Rotate Region from the Gates
menu by holding down the Command key while dragging a region handle.
You can stretch, reposition, and rotate rectangular and elliptical regions in the
same way you did the polygonal region.
Repositioning Vertices
Another method of adjusting a polygonal region is to reposition the vertices. To do this,
double-click on the region border to put it in edit mode; or click once on the region to
select it, and choose Edit Polygon from the Gates menu. You will see handles at each of
the vertices. Click and drag these handles to reshape the region.
When you have finished, choose Stop Editing Polygon from the Gates menu, or simply
click on the plot anywhere outside the region.
1 Double-click the border of region R1.
Handles will appear at each vertex of the region.
Vertex
Handles
vertex
handles
BD CellQuest Pro Software
337452 Rev. A
123
2 Drag the vertices to make region R1 encompass the monocyte cluster.
3 Reposition the region R1 vertices in their original positions.
4 Click outside the region.
Showing and Hiding Regions
You can hide a region outline from view in a plot. A hidden region remains in the
Region list, where it can be copied and pasted into other plots.
1 Select region R1 if it is not already selected.
2 Press Option-Delete simultaneously.
The R1 region outline disappears from the plot.
3 Press the Command key (a) while clicking inside the plot.
A pop-up menu appears.
4 Select R1 from the pop-up menu.
The R1 region outline reappears in the plot.
124
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Deleting a Region
1 Draw a region around the monocytes in the FSC vs SSC plot (R2).
2 Make sure region R2 is selected, then press Delete.
The region is removed from the plot and the Region List. To delete multiple
regions in a plot, click each region while holding down the Shift key, then press
Delete. Alternatively, you can choose Edit > Cut or Clear.
Using Snap-To Regions
Use the Snap-To–Region tool to automate cluster analysis in a dot, density, or contour
plot. After you click on a distinct cluster, the Snap-To–Region tool automatically draws
a region around the population. When a new data file is read into the plot, the region
automatically redraws around the new population.
1 Click the Snap-To–Region tool (
) in the tool palette.
2 Click on the monocyte population in the FSC vs SSC dot plot.
A region is automatically drawn. Note that the region outline appears thicker
than that of other regions to differentiate the Snap-To feature.
A Snap-To region can be edited just like a polygonal region. Click and drag any of
the handles to resize the region and click on the border of the region to drag it to
a new position.
If the cluster is too small or diffuse for the Snap-To–Region tool to define a
region, the system beeps and no region is drawn. In this case, you might have to
use another of the region tools to draw the region.
3 Select the FSC vs SSC plot and choose Select File from the File pop-up menu in
the Inspector.
A dialog box appears.
BD CellQuest Pro Software
337452 Rev. A
125
4 Go to BD Apps/CellQuest Pro/Sample Files and open data file NORM001.
Notice the R2 Snap-To region has been redrawn. A Snap-To region is
automatically redrawn when the data in the plot changes because the plot’s gate is
changed or a new data file is read into the plot.
5 Duplicate the FSC vs SSC plot.
Select the plot and choose Edit > Duplicate. A new plot appears. Notice that the
duplicate plot does not display region outlines.
6 Display the region outlines in the new FSC vs SSC plot.
Hold down the Option key and click anywhere inside the plot. Choose Show All
Regions from the pop-up menu that appears.
7 Move R2 in the new plot so that the region excludes part of the monocyte
population.
8 Select both FSC vs SSC plots and choose Select File from the File pop-up menu in
the Inspector.
9 Open NORM002.
Notice that the R2 Snap-To region in the original FSC vs SSC plot redraws. The
region in the duplicate FSC vs SSC plot also changes because it is the same as the
region in the original FSC vs SSC plot.
10 Delete the duplicate FSC vs SSC plot.
Select the plot and press Delete or choose File > Cut or Clear.
11 Delete all regions.
Select the regions and press Delete.
126
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Using Other Snap-To Region Features
You can do one or more of the following with Snap-To regions.
•
Adjust Auto Movement setting
•
Adjust Auto Size setting
•
Tether to other regions
To practice using these enhanced features, do the following.
1 Create a new Experiment document.
Choose File > New Document.
2 Choose Help > CellQuest Pro Help.
3 Enter Snap-To Regions in the search field and press Enter.
Ask a Question field
4 Double-click Becoming Familiar With Snap-To Regions.
BD CellQuest Pro Software
337452 Rev. A
127
5 At the end of the tutorial, click the right arrow on the navigation bar to move to
the next tutorial, Tethering Snap-To Regions.
You can print the results of each tutorial, if you want.
Using Region Stats to Analyze Data
The following exercise demonstrates how regions and region statistics can be used to
get population statistics.
1 Verify the Gating Exercise Experiment document is opened.
2 Display data file 3C.005 in all plots.
•
Select the plots and choose Select File in the Inspector.
•
Go to the Training Files/Gating folder and open 3C.005.
3 Set a polygonal region around the lymphocyte cluster on the FSC vs SSC plot.
Region R1 is drawn.
4 Set an elliptical region around the monocyte cluster.
Region R2 is drawn.
5 Set a rectangular region around the granulocyte cluster.
Region R3 is drawn.
6 Use the techniques you learned in the previous exercises to stretch and rotate
these regions to conform to the clusters.
7 Select the dot plot and choose Stats > Region Stats.
128
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Questions
1 Review the statistics and record the % Lymphs, Monocytes, and Granulocytes
present in this sample.
Lymphocytes __________________
Monocytes ____________________
Granulocytes __________________
NOTICE These results are only estimates and should never be used in place of a
hematology differential.
2 Why were the statistics listed under % Gated and % Total the same?
_____________________________________________________________________
3 Create a region and view the region statistics to determine the % CD3–CD4+ cells
on the FITC vs PE plot. Record your result.
% CD3–CD4+ cells _____________
When you finish these questions, delete any statistic views from the document.
Using Gates
A gate consists of one or more combined regions. Use gates to isolate subsets of data for
analysis or sorting. By default, BD CellQuest Pro software assigns gate 1 to region 1
(G1 = R1), gate 2 to region 2 (G2 = R2), and so on. You can modify these assignments
according to your experiment.
Backgating (Fluorescence Gating)
Backgating is useful for determining where a population of interest on one plot is
located on another plot. Backgating is done by drawing a region around a population
on a fluorescence plot, and using this region as a gate to view the population on another
plot (usually FSC vs SSC). It is called backgating because you view the fluorescence plot
before viewing the scatter plot (usually it’s the other way around). The following
exercise will illustrate this point.
BD CellQuest Pro Software
337452 Rev. A
129
1 Draw a region (region 4) around the CD3–CD4+ population on the FITC vs PE
plot if you have not already done so.
2 Select the FSC vs SSC plot and then choose G4 = R4 from the Gate pop-up menu
in the Inspector.
You are now viewing the scatter properties of events that fell within the R4
region.
Questions
1 Based on the scatter properties of the events within R4, what cell type makes up
the CD3–CD4+ population?
_________________________________________________________
130
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
2 Use backgating to determine the location of the basophils on the FSC vs SSC plot.
Hint: The location of the basophils is shown below on the FL3 vs SSC plot.
basophils
What population does the basophil population resemble on an FSC vs SSC plot?
_____________________________________________________________________
Using Multicolor Gates
Multicolor gating allows you to see populations that qualify for a gate without actually
gating the data. The following exercise demonstrates this feature.
1 Select all plots in the Experiment document.
2 Click the Multicolor Gating checkbox under Dot Plot and choose No Gate from
the Gate pop-up menu in the Inspector.
The dot plots now show the location of the lymphocyte data (red), monocyte data
(green), and granulocyte data (magenta). As shown in step 5, these colors are
associated with gates, not regions.
3 Choose Gates > Gate List.
On the Gate List, under the Color heading, you will see the colors associated with
each gate. See Figure 4-14 on page 132.
BD CellQuest Pro Software
337452 Rev. A
131
Figure 4-14 Gate List dialog
4 Click the red square under Color next to the G1 label.
The Select gate color dialog box appears.
5 Click the yellow rectangle.
The color box next to G1 is now yellow.
6 Click the close box in the upper left corner of the Gate List dialog box.
Note that the lymphocyte data is now colored yellow.
7 Repeat steps 4 through 6, but this time return the color of Gate 1 to red.
Reordering the Gate List
Events in gates are colored in the order the gates appear in the Gate List. You can alter
the order in which events are colored by simply reordering the gate list. Here’s an
example of why you might want to do this.
1 If you have not drawn a region (region 5) around the basophils on the FL3 vs SSC
plot, create one now.
2 View the FL3 vs SSC display and note the basophils are colored red even though
the color for G5=R5 is rust.
Data points are colored according to the order in which the gates appear on the
Gate List. Therefore, all data points that qualify for gate 1 are colored red. Next,
any data points that qualify for gate 2 and have not already been colored are
colored green, and so on for each gate color. Since basophils fall within R1, they
are colored red.
3 To display the basophils as rust colored, move G5 to the top of the Gate List.
Choose Gates > Gate List, and then click on the selection arrow by G5 and drag
the G5 line to the top of the list.
132
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 View the FL3 vs SSC plot and note that the basophils are now colored rust.
The events in G5 are colored first. These events will not be colored red even
though they fall in G1 because they have already been colored with the G5 color.
5 View the FSC vs SSC plot.
Question
1 Notice that it is difficult to distinguish the rust-colored events from the red events
because the colors are similar. What could you do to better visualize the basophils
against the lymphocyte population on the FSC vs SSC plot? Try it.
_____________________________________________________________________
Click Set All to Default on the Gate List before you proceed with the next exercise. This
will restore the default gate colors and gate order to the Gate List.
Using Combined Gates
Regions can be combined to create more complex gates for analysis and sorting. There
are three logical operators—and, or, and not—used to combine regions. You can use the
words or their equivalent symbols in gate definitions.
R1 AND R2
(R1*R2)
When the AND (*) operator is used, only events that are in both
regions satisfy the gate.
Be careful when using AND. If you wanted a gate that would show
you the fluorescence of lymphocytes and granulocytes on a scatter
plot, using AND to combine lymphocyte and granulocyte regions
would not work since no cells fall in both regions. You would use the
OR operator for this gate.
R1 OR R2
(R1+R2)
When the OR (+) operator is used, an event that falls in either
region satisfies the gate.
NOT R1
(–R1)
When the NOT (–) operator is used, any event that falls
outside the region satisfies the gate.
You can begin an expression with NOT, for example, NOT R1
AND R2. However, if NOT appears in the middle of the
expression, it must be preceded by an operator.
R1 AND NOT R2 is a valid expression; R1 NOT R2 is not.
When defining gates, be sure to leave spaces between the region designations and the
AND and OR operators.
BD CellQuest Pro Software
337452 Rev. A
133
The logical operators are applied in this order: NOT, then AND, then OR. You can use
parentheses to change this order. The operations and regions in parentheses are read
first, before the other operators.
Following are three examples.
•
NOT R1 AND R2. CellQuest Pro first reads NOT R1, then AND R2: all events
outside R1 and also in R2.
NOT (R1 AND R2). CellQuest Pro first reads R1 AND R2, then applies NOT: all
events outside the intersection of R1 and R2.
•
R1 OR R2 AND R3. CellQuest Pro first reads R2 AND R3, then R1 OR: all
events in both R2 and R3 or events in R1.
(R1 or R2) AND R3. CellQuest Pro first reads R1 OR R2, and then AND R3: all
events in either R1 or R2 and also R3.
•
R1 OR G2 OR NOT R3 AND R4. CellQuest Pro first reads NOT R3, then reads
AND R4, and then R1 OR G2.
(R1 OR G2 OR NOT R3) AND R4. CellQuest Pro first determines the events
within the parentheses using the normal order of operations, then it reads AND
R4.
You can use a logical gate in the definition of another logical gate, for example, R1 and
G2. You can use the same region or logical gate any number of times in a logical gate
definition. You can copy regions from one Experiment document to another, but you
cannot copy gates.
To practice using combined gates, you will define a gate to capture all the CD4+ events.
1 Make sure all plots in the document are displaying all events (No Gate is applied).
2 On the FL1 vs FL2 plot, draw region 6 around the CD3+CD4+ population and
region 7 around the CD3+CD4– population.
Be sure to extend R7 to the baseline so you do not miss any events.
3 Choose Gates > Gate List and define G8 as R4 OR R6.
Be sure to leave spaces between the region designations and the OR operator.
4 Press the Return key.
The definition you typed should change from italic letters to non-italic. This
indicates that you have specified a valid gate.
5 Close the Gate List.
134
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
6 Activate the FL1 vs FL2 plot and choose Stats > Gate Stats.
Note the % Total statistic for G8: _______
You have just calculated the % CD4+ cells (monocytes and lymphocytes) in the
sample.
7 Make sure the FL1 vs FL2 plot is selected and the Dot Plot Inspector is open;
choose G1 = R1 from the Gate pop-up menu in the Inspector.
View the FL1 vs FL2 plot and the Gate statistics.
8 Note that the % Gated statistics are now different from the % Total statistics.
The % Gated statistics are calculated by dividing the number of events in a gate
by the number of events in the gate used to create the display (G1); the % Total
statistics are calculated by dividing the number of events in a gate by the total
number of events in the data file.
9 Change the FL1 vs FL2 plot back to No Gate.
Select the FL1 vs FL2 plot and choose No Gate from the Gate pop-up menu in the
Inspector.
10 Choose Gates > Gate List and change the definition of G8 to R4 AND R6.
After you enter the definition, don’t forget to press Return.
11 View the Gate stats and note the % Total statistic for G8: __________
The G8 % Total statistic is zero because no events satisfy both R4 and R6 since
they are two different populations. Now, let’s try a more complicated gate.
12 Change the definition of G8 to R1 AND NOT (R5 OR R6 OR R7).
13 View the Gate stats.
The G8 % Total statistic reflects lymphocytes that are not CD3+CD4– or
CD3+CD4+. In other words, these are the non-T lymphocytes in the sample.
Questions
1 Is the statistic for G8 (%Gated or %Total) the percentage of lymphocytes that are
non–T lymphocytes, or the percentage of all cells that are non–T lymphocytes?
_____________________________________________________________________
BD CellQuest Pro Software
337452 Rev. A
135
2 Describe what you could do to view the scatter of the non–T lymphocytes in
comparison with all lymphocytes. Try it.
_____________________________________________________________________
3 Write an expression to define a gate that contains events that are not granulocytes
and are CD4+. Enter this expression as the definition for G8 in the Gate List.
_____________________________________________________________________
4 Gate the FSC vs SSC plot using G8. If your definition from question 3 is correct,
you should see some lymphocytes and all the monocytes on the scatter plot. If it is
not correct, redo you definition until the correct populations appear on the gated
scatter plot.
5 Write an expression to define G8 as a gate that contains all non-lymphocytes that
are CD4+. Enter this expression in the Gate List.
_____________________________________________________________________
6 Record the % Total statistic for G8.
_____________________________________________________________________
7 Use G8 as a gate to view the FSC vs SSC characteristics of the population. Based
on scatter, what population did you define with G8?
_____________________________________________________________________
8 Activate the FSC vs SSC plot and choose Gate Stats from the Stats menu. Explain
why the % Gated and % Total statistics are different.
_____________________________________________________________________
_____________________________________________________________________
Using a Combined Gate for Histogram Analysis
Combined gates can also be used to gate data for quadrant analysis. In this exercise,
you will combine two regions to create a lymphocyte gate.
1 Delete all regions from the region list and clear all stats views from the document.
2 Set All to Default in the Gate List window.
3 Change all plots to display ungated data.
Select all plots and then choose No Gate from the Gate pop-up in the Inspector.
136
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 Change the X parameter of the FSC vs SSC dot plot from FSC to FL1
(CD3 FITC).
You can do this either by clicking on the parameter label on the plot and selecting
another parameter from the pop-up menu that appears or by selecting the plot
and choosing a parameter from the Parameter pop-up menu in the Inspector.
5 Create a region (R1) around the lymphocyte cluster in the FL3 (CD45 PerCP) vs
SSC plot.
6 Create a region (R2) around the CD3+ cells in the FL1 (CD3 FITC) vs SSC plot.
Defining a Gate
Next you will combine regions 1 and 2 into a single gate that defines CD3+ lymphocytes
using three parameters: SSC, CD45, and CD3.
1 Choose Gate List from the Gates menu.
2 Define G3 as R1 AND R2.
BD CellQuest Pro software allows you to label gates. Before you exit the Gate
List, you can label the gate you just created.
3 Double-click the Label field that reads G3.
G3 becomes highlighted.
BD CellQuest Pro Software
337452 Rev. A
137
4 Enter CD3+ Lymphs as the Gate Label.
5 Close the Gate List window.
Viewing Gate Statistics
1 Next, you will use the gate you just created to get histogram statistics on the
lymphocyte population.
2 Click the Histogram Plot tool in the tool palette and use it to create a histogram
plot.
3 Choose Analysis as the Plot Type, click Select File, and open data file 3C.005.
Data file 3C.005 is located on the hard drive in the Training Files folder, then in
the Gating folder.
4 Make sure that the checkbox next to Manual Scale in the Histogram section of
the Inspector is selected.
5 Change the Manual Scale setting to 50.
Manual Scale setting
6 Choose FL2 as the X parameter, then choose CD3+ Lymphs in the Gate pop-up
menu in the Inspector.
138
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
7 Click the histogram marker tool (
) on the tool palette.
8 Draw a histogram marker to delineate the negative cells on the FL2 histogram.
Draw a second marker to delineate the positives.
Normally you would set markers such as these using an appropriate control.
9 With the FL2 histogram active, choose Stats > Histogram Stats.
Record the % Gated and % Total statistics for the positive cells.
M2
% Gated _____________ % Total _____________
Questions
1 Which statistic reflects the % of CD3+ lymphocytes that are CD4+? (Check one.)
M2
% Gated _____________ % Total______________
2 What percentage of CD3+ lymphocytes are not CD4+ cells? __________________
BD CellQuest Pro Software
337452 Rev. A
139
140
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Optimization
Three- or Four-Color Immunophenotyping
337452 Rev. A
Big Picture
Start Up
System
•
•
•
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Set up a BD CellQuest Pro Experiment document.
Optimize instrument settings for your sample type.
Save optimized settings.
2
1
Optimization Steps
1. Check FSC & SSC settings.
2. Check FSC threshold setting.
3. Gate population of interest.
4. Check fluorescence PMT settings.
5. Check compensation settings.
3
Samples for Optimization
• Isotype control (γ1 FITC/γ1PE/ γ1PerCP/γ1APC)
• FITC control (CD3 FITC)
• PE control (CD8 PE)
• PerCP control (CD45 PerCP)
• APC control (CD4 APC)
4
2
Types of Binding
antigen-specific
binding
non-antigen−specific
binding
non-specific
binding
5
Overcompensation vs
Undercompensation
Correct
Compensation
Overcompensation
Undercompensation
10%
30%
30%
20%
6
3
Adjust FSC & SSC Settings
neutrophils
monos
lymphs
Pre
Post
7
Adjust FSC Threshold
debris
Pre
Post
8
4
Gate Population of Interest
R1 around Lymphocytes
9
Optimize Fluorescence PMT Settings
Plots are gated to display R1 events.
Pre
Post
10
5
Optimize Compensation – FITC Control
FL2-%FL1
Pre
Post
11
Optimize Compensation – PE Control
FL1-%FL2
FL3-%FL2
Pre
Post
12
6
Optimize Compensation – PerCP
Control
FL2-%FL3
FL4-%FL3
Pre
Post
13
Optimize Compensation – APC Control
FL3-%FL4
Pre
Post
14
7
Samples to Acquire
• PerCP control
• CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC
• CD3 FITC/CD16+CD56 PE/CD45 PerCP/CD19 APC
15
8
Reagent List
CD3
CD4
CD8
CD14
CD16
CD19
CD45
CD56
Pan T Lymphocytes
Helper T cells, Monocytes
Suppressor/Cytotoxic T cells, Natural Kiler (NK) cells
Monocytes
Resting NK cells
Pan B cells
Pan Leukocytes
Resting and activated NK/T cells
Phenotypes
CD3+CD4+
CD3–CD4dim
CD3+CD8+
CD3+CD16/CD56+
CD3–CD16/CD56+
Helper T cells
Monocytes
Suppressor/Cytotoxic T cells
Cytotoxic T cells
NK cells
337452 Rev. A
337452 Rev. A
Icons
How to Save
Choose File > Save Document.
Deselect the Setup checkbox in
the Acquisition Control window
or in the Acquisition Browser.
Choose Cytometer > Instrument
Settings. Click Save.
Click the Export statistics
checkbox in the Batch Setup
window. You can also save an
export file by choosing File >
Export Statistics.
Description
An Experiment document contains
plots, regions, quad markers, gates,
acquisition setup, and so on.
An FCS data file contains raw data
acquired from the cytometer. It also
contains instrument settings used at
the time data was collected. Settings
can be retrieved and used for other
experiments, if needed.
An instrument settings file contains
PMT voltage, Amp Gain,
Compensation, and Threshold
settings.
An export file is a text file containing
statistical values, eg, quadrant stats,
that can be used to transfer data into
other applications such as a
spreadsheet or database.
Launch a spreadsheet
application, eg, Microsoft®
Excel, choose File>Open, and
select the export file to open.
Choose Cytometer >
Instrument Settings. Click
Open. Select the settings file,
click Open, then click Set. Click
Done.
Select an analysis plot, choose
Select File from the File pop-up
menu in the Inspector.
Choose File > Open Document
or double-click the document
icon.
How to Open
BD CellQuest™ Pro File Types
5
BD MultiSET Software
After completing this module, you will be able to:
•
Optimize and save instrument settings using the Optimization view in
BD FACSComp software.
•
Acquire and analyze three- or four-color lyse/no-wash samples prepared in
BD TruCOUNT™ tubes.
•
Use BD MultiSET™ software tools and preferences to customize acquisition and
analysis.
•
Use BD MultiSET software user-defined features.
BD MultiSET Software
337452 Rev. A
141
Acquisition of data using BD MultiSET software is a multistep process. The following
figure outlines the main steps involved. In this module, we will focus on QC,
optimization of instrument settings, data acquisition, and data analysis.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
In the following exercises, you will:
•
Perform instrument QC in BD FACSComp software
•
Optimize instrument settings
•
Define conditions for data acquisition and storage
•
Acquire and analyze data
You will run BD Multi-Check control and peripheral blood samples stained with
BD MultiTEST™ or BD TriTEST™ reagents and prepared in BD TruCOUNT™ tubes.
Refer to the appropriate reagent product insert for details.
The BD Multi-Check Control is intended as a complete process control for
immunophenotyping by flow cytometry. It is a control for antibody staining, red blood
cell (RBC) lysis, instrument setup and performance, and data analysis. It is a stable
control with assigned values that can be used to monitor the immunophenotyping
process. The control should be treated in the same manner as whole blood. Refer to the
BD Multi-Check control package insert for details.
BD TruCOUNT tubes can be used to determine absolute counts of lymphocyte
subpopulations. Each tube contains a known number of beads in a lyophilized bead
pellet. Refer to the BD TruCOUNT tube package insert for details.
NOTICE The following exercises can be used to acquire and analyze samples stained
with BD TriTEST or BD MultiTEST reagents and prepared in BD TruCOUNT tubes. If
you are not using BD TruCOUNT tubes, skip the steps related to BD TruCOUNT
tubes.
Before performing the exercises in this module, start up the BD FACSCalibur system by
following the System Startup on page 20 in the BD FACSCalibur System module.
142
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Performing QC
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
In Module 2 you launched BD FACSComp software from the Dock. In this exercise you
can will launch the software from within BD MultiSET software.
In this exercise you will:
•
Complete the Sign In view in BD MultiSET software.
•
Complete the Set Up view in BD MultiSET software.
•
Launch BD FACSComp software from within BD MultiSET software and
perform instrument QC.
Sign In View
1 Launch BD MultiSET software from the Dock.
The Sign In view appears.
2 Enter an Operator name.
The operator ID is required. The information at this view will be saved for
subsequent sessions.
BD MultiSET Software
337452 Rev. A
143
3 Click Accept.
The Set Up view appears.
Set Up View
In this view, you will make selections under Data Source, Entry Level File Name Prefix,
View Reports, and Automatic Savings Options.
•
When From Cytometer: Acquisition with Analysis radio button under Data
Source is selected, analysis occurs automatically after samples are acquired. This
is referred to as cytometer-based acquisition.
•
When the Sample Name radio button under Entry Level File Name Prefix is
selected, the file name prefix for the FCS data files, Laboratory Report file, and
Physician Report file are named according to information entered in the Sample
Name column in the Samples view.
•
When the Until “Next” Button Pressed radio button is selected under View
Reports, you can view the analysis display for as long as you like.
•
When the Automatic Saving Options boxes are checked, the Laboratory,
Physician, and Summary Reports, and Export files are saved.
1 Verify the following selections are made under in the Set Up view.
current file path
Note that the current path appears below each file type. The default location for
each file type is a dated folder (DDMMYY), in the BD Files/MultiSET Files folder.
144
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
2 Click Accept.
The FACSComp view appears because the data source is From Cytometer.
NOTICE The Test Prefs view appears (Figure 5-2 on page 150) if the data source is
From Data Files.
The name of the most recent Calib file is displayed along with the date and time
the file was modified as well as whether it passed or failed.
CAUTION These calibration settings are not automatically downloaded to the
cytometer.
BD FACSComp Software
It is assumed that you are already familiar with how to perform instrument QC using
BD FACSComp software. If not, see the BD FACSComp Software module for details.
1 Click Launch FACSComp.
BD FACSComp software launches and the Setup view is displayed.
2 Select the Lyse/No-Wash (LNW) option under Assay Selections. Verify none of
the other options are selected.
3 Prepare appropriate BD CaliBRITE beads and perform instrument QC.
Instrument settings are saved to the Calib File.LNW if all parameters pass
sensitivity testing.
BD MultiSET Software
337452 Rev. A
145
Optimizing Settings
In this exercise, you will optimize instrument settings as outlined in the following
graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Optimize SSC setting, if needed.
• Optimize FL3 threshold setting, if needed.
• Save optimized settings.
Optimizing SSC Setting
The instrument settings generated by BD FACSComp software after the LNW setup
option is run are appropriate for LNW samples. However, you still might need to
optimize the SSC and FL3 threshold settings.
In this exercise, you’ll practice performing optimization using the Optimization view
provided in BD MultiSET software.
The Optimization view provides three predefined dot plots you can use to view live
events, without saving data, as you adjust settings. Once optimization is complete, you
can save your optimized settings.
1 At the Summary Report view, click Optimization.
This view allows you to fine tune instrument settings, if necessary, before
acquiring data.
pop-up menu
146
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Up to five optimized instrument settings files—Optimization 1 through
Optimization 5—can be listed in the pop-up menu. A default file name is
associated with each optimization option. To change the name, use the
Optimization Preference dialog box. Refer to the BD FACSComp Software User’s
Guide for details.
The initial option displayed is determined by the Optimization Option selected in
Optimization preferences. You can change the display at this view to any of the
five options by choosing it from the pop-up menu located below the left-most
plot.
2 Click the pop-up menu, and choose the appropriate optimization file name.
The plot parameters change to reflect the new option, and the instrument settings
are downloaded to the cytometer.
NOTICE You can also read any other instrument settings file by using the
Instrument Settings dialog box accessed through the Cytometer menu.
3 Install any of the control or sample tubes on the cytometer, and click Start.
You can change the plot parameters by clicking on the axis label and choosing a
parameter from the pop-up menu.
4 Adjust the SSC PMT if necessary.
Data displayed in the FSC vs SSC plot after the SSC PMT setting is optimized
should look similar to that shown in Figure 5-1 on page 148.
BD MultiSET Software
337452 Rev. A
147
Figure 5-1 Optimization view
CAUTION Do not modify the fluorescence PMT detector or compensation settings.
5 Adjust the FL3 threshold to exclude most of the debris.
Adjust the FL3 threshold level until the majority of the debris is removed without
cutting off any of the lymphocyte population. A small amount of debris should be
present for the Expert Gate to set properly. If running both BD TriTEST and
BD MultiTEST samples, optimize once with BD MultiTEST samples.
NOTICE If your BD TriTEST panel includes both CD4/CD8/CD3 and
CD3/CDX/CD45, optimize using the CD4/CD8/CD3 tube.
6 Click Save Optimized Settings.
A file name and location dialog box appear if instrument settings have been
previously saved with the default name. Click Overwrite to overwrite the file with
the current optimized settings.
7 Click Quit.
The FACSComp view in BD MultiSET software appears.
8 Click Continue.
The Test Prefs view appears. See Figure 5-2 on page 150.
148
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Acquiring Data
In this exercise, you will acquire and analyze data as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Specify data collection information (file name, storage location, panels, and so on).
• Click Acquire.
• Gate manually, if needed.
• Print or export results, if needed.
You will set up BD MultiSET software to acquire a control sample (BD Multi-Check
control) and a mock patient sample. Samples provided during the training week are
whole blood stained with one of the following reagent combinations.
Three-Color Panel
•
CD3 FITC/CD4 PE/CD45 PerCP
•
CD3 FITC/CD8 PE/CD45 PerCP
•
CD3 FITC/CD16+CD56 PE/CD45 PerCP
•
CD3 FITC/CD19 PE/CD45 PerCP
Four-Color Panel
•
CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC
•
CD3 FITC/CD16+CD56 PE/CD45 PerCP/CD19 APC
Specifying Data Collection Information
In this exercise, you select what you want displayed on the Laboratory, Physician, and
Summary Reports. You will also enter information for the BD TruCOUNT tubes. If you
are not using BD TruCOUNT tubes when you return to your lab, it is not necessary to
enter BD TruCOUNT tube lot ID and beads/pellet information.
Test Prefs View
In this view, you will make selections under Physician Report Choices, Laboratory
Report Choices, and Summary Report ID. You will also access the Lot IDs dialog to
enter BD TruCOUNT bead information.
•
All subsets for BD-defined reagents are listed under Physicians Report Choices.
Selected subsets will be included on the Physicians Report if reagents producing
these results are part of the entry’s panel.
BD MultiSET Software
337452 Rev. A
149
•
Reference ranges appear on the Physicians Report when the Report Reference
Ranges checkbox is selected. Reference ranges are defined within the Subset
Ranges dialog. The range selected in Panel Tools is the range applied to the
Laboratory and Physicians Reports.
•
Percentages and absolute counts are reported on the Laboratory Report when
Report Percents and Report Absolute Counts under Laboratory Report Choices
are selected. In addition, the information is reported on the Physicians Report.
•
The choices made under Summary Report ID determines what appears on the
Summary Report for each entry.
1 Verify the following are selected in the Test Prefs view.
Figure 5-2 Test Prefs view
2 Click Lot IDs.
3 Click the Absolute Count Beads icon.
4 Enter the Lot ID and Beads/Pellet information found on the BD TruCOUNT tube
foil pouch label.
These values are found on the BD TruCOUNT tube foil pouch label. This
information is critical for calculating absolute counts. Only one lot ID can be
entered per Schedule document.
150
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
5 Click Save.
6 Click Accept to continue to the Samples view.
Samples View
You must enter information in the ID column associated with the entry-level file name
prefix, which you selected in the Set Up view. In this exercise, that is the Sample Name.
This prefix is used for naming data files, Laboratory Report files, and Physician Report
files. Also, enter information in the field(s) selected in the Test Preferences view for the
Summary Report ID. In this exercise that is also the Sample Name. The message box at
the top of the window will indicate which fields are required.
NOTICE When using BD TruCOUNT tubes, do not enter information in the WBC or
Lymph fields. If you are not using BD TruCOUNT tubes when you return to your lab, it
is not necessary to enter BD TruCOUNT tube lot ID and beads/pellet information.
Enter either a WBC and Lymph (%) obtained from a hematology instrument, or an
absolute lymphocyte count (x1000) obtained from a hematology instrument. You
cannot enter both.
1 Click the Sample Name field in row one, and enter a name for the control sample.
2 Click and hold in the Panel Name column and choose the 3C TBNK + TruC or
4C TBNK + TruC Panel from the pop-up menu.
3 Enter a Sample Name for the second sample.
4 Choose the 3C TBNK + TruC or 4C TBNK + TruC Panel from the pop-up menu
for the second sample.
BD MultiSET Software
337452 Rev. A
151
Retrieving Instrument Settings
To practice retrieving saved instrument settings, do the following to download the
instrument settings you saved in the previous section.
1 Choose Cytometer > Instrument Settings.
2 Navigate to the BD Files/Instrument Settings Files folder and double-click the
TriTEST.opt or MultiTEST.opt settings to open the file.
This is the optimized instrument settings file you created in BD FACSComp
software. The Instrument Settings dialog box is available from the Cytometer
menu at the Samples view, Lab Report view, Physician Report view, and Summary
Report view. If you run samples requiring different optimized instrument settings,
download these settings from the Physician Report view between panels.
NOTICE You can download a TriTEST.opt or MultiTEST.opt FACSComp
instrument settings file for a TriTEST three-color application.
3 Click Set. Click Done.
Saving a Schedule Document
A BD MultiSET software Schedule document file can be saved. The file contains all data
information entered at the Sign In, Set Up, Test prefs, and Samples views, as well as
preferences. The file does not contain information entered in Subset Ranges, Panel
Tools, Reagent Tools, and Lot IDs.
A saved Schedule document can be opened later to view, to complete processing, or to
add more samples for processing. Only one Schedule document can be open at a time.
You cannot print a Schedule document.
1 Choose File > Save.
2 Enter a name for the file and choose a folder destination.
During the training class, choose your folder in the Student folder as the
destination.
3 Click Save.
152
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Sample Acquisition and Analysis
The Acquisition view allows you to view a live display of samples before and during
acquisition. Data is not saved until you click Acquire. Once a sample has been run on
the cytometer, the sample information on the Samples view can no longer be edited.
Acquisition continues until the designated number of events to acquire is reached.
BD MultiSET software performs an initial acquisition by acquiring the number of
events set in the Events Preferences. The software then checks to see if the minimum
target population events have been collected. The acquisition target events are set in
Reagent Tools. If the target population event numbers have been reached, BD MultiSET
software proceeds to analysis. If the target population event numbers have not been
reached, the software calculates the additional number of events needed to satisfy the
target population requirement.
1 Vortex the first control tube, and install it on the cytometer.
2 Verify the RUN and HI flow rate buttons are selected.
3 Click Run Tests.
4 Click Acquire.
The acquisition phase begins.
BD MultiSET Software
337452 Rev. A
153
Lab Report View
The Laboratory Report view appears when acquisition for a single tube is complete. As
each tube within a panel is acquired, the information is added to the Laboratory
Report. When appropriate, QC Messages are reported. The Laboratory Report file is a
PDF file that can be opened and printed by Adobe® Acrobat® Reader or any software
that reads PDF files.
1 Remove the tube from the cytometer and install a tube of DI water on the SIP.
2 Place the cytometer in standby mode.
attractor gate
expert lymph gate
Because Until “Next” Button Pressed was selected in the Set Up view, you can
view the report as long as you want.
Manual Gating
If needed, manual adjustments to the expert lymph gate and the attractors can be made.
In this exercise, you will practice using this feature.
The expert lymph gate identifies the lymphocyte population from a CD45 vs SSC or
CD3 vs SSC display. Attractor regions then single out lymphocyte subsets from
fluorescence parameter combinations. Attractors are able to automatically adapt
154
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
themselves to position shifts of populations. Refer to the BD MultiSET Software User’s
Guide for details about the expert lymph gate. Refer to the BD Attractors Software
User’s Guide for details about attractor regions.
You will adjust the expert lymph gate and other gates to practice manual gating.
1 Click Manual Gate.
You can adjust the gate or the attractors for the tube just analyzed. Any changes
made to the gate, attractors, or display are applied to the current tube only.
2 Double-click the CD45 vs SSC plot.
The plot zooms to a larger size.
3 Position the cursor at a starting point near the lymphocytes and click.
This is the starting point for the gate.
4 Continue clicking until a polygon surrounds the lymphocytes.
5 Click the close box in the upper left corner of the plot to un-zoom the plot.
6 Practice adjusting the attractors.
Attractor gates might occasionally need adjustments.
You can move, resize, and reorient the attractors, but you cannot delete them or
add new ones.
•
To move an attractor, click and drag the center point into position.
BD MultiSET Software
337452 Rev. A
155
•
To change the orientation and length, drag one of the two endpoints.
•
To modify the size of an attractor, drag an edge
7 Click Analyze.
This confirms the changes and recalculates the results. A message appears in the
QC Message area of the Lab Report indicating that a manual gate has been set.
8 If needed, save the changes you made.
You can save user-defined changes made to the attractors. Changes made to the
expert lymph gate are not saved. To practice saving the changes:
•
Choose File > Save Reagent.
•
Enter a unique name, or keep the default name in the dialog box that
appears.
•
Click OK.
Once saved, the new user-defined reagent can be used again the next time a tube
with the same reagent combination is analyzed. The name of the reagent will have
UD appended to it to indicate that it is user-defined. Refer to the BD MultiSET
Software User’s Guide for details about user-defined reagents.
9 Vortex and then install the next control tube on the SIP. Verify the RUN button is
selected.
10 Click Continue.
The software proceeds with acquisition and analysis of the next tube.
11 After this second control tube has been acquired, install a tube of DI water on the
SIP, and place the instrument in standby mode.
Since this is the last tube in the panel, entry-level QC values are displayed on the
Lab Report. See Figure 5-3 on page 157.
156
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 5-3 Entry Level QC in Laboratory Report view
NOTICE After all tubes in the panel have been run and before proceeding to the
Physician Report view, you can rerun any of the tubes in the panel.
12 Click Continue.
The Laboratory Report prints if this was selected in the printing preferences.The
Physician Report appears. See Figure 5-4 on page 158.
Physician Report View
The Physician Report is a summary of information for one sample. Figure 5-4 on
page 158 provides an example. The report contains all results that were selected for
each reagent in a panel. If more than one reagent in a panel provides a particular subset
result, the average subset result is reported.
BD MultiSET software reports multitube results, including lymphosum, two CD3
consistency results (% CD3 and CD3 Abs Ct Range), and a T-helper/suppressor (T H/S)
ratio determined from more than one tube.
The report also contains a Comments section where you can enter the lab director’s
name as well as any additional sample information. The Verbal Order checkbox allows
you to indicate when a test was verbally requested by a physician. The Physician Report
file is a PDF file that can be opened and printed by Adobe Acrobat Reader or any
software that reads PDF files.
BD MultiSET Software
337452 Rev. A
157
Figure 5-4 Physician Report view
Reviewing Results
You will review your results from the control sample and compare the values with those
in the Assay Values insert included with the BD Multi-Check control.
158
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
1 Enter your results in the space provided.
For CD3+ cells, enter the average percentage and absolute count from the two
tubes.
Population
Percentage
Absolute Count
CD3+ cells
CD3+/CD4+ cells
CD3+/CD8+ cells
CD3+/CD16+CD56+ cells
CD3+/CD19+ cells
2 Compare your results with those listed on the Assay Values insert.
NOTICE Each laboratory needs to establish its own acceptance criteria.
Acquiring the Remaining Samples
1 Install the first sample tube on the SIP.
2 Verify the RUN and HI flow rate buttons are selected.
3 Click Next at the Physician Report view.
The Physician Report prints if this was selected in the preferences.
4 Click Acquire.
5 Install the second sample tube when appropriate. Click Continue.
6 After this second sample tube has been acquired, install a tube of DI water on the
SIP, and place the instrument in standby mode.
7 Click Continue. Click Next.
Because this was the last entry in the Schedule document, the Summary Report
view appears. See Figure 5-5 on page 160. The Summary Report contains a list of
all entries in the Schedule document. The view has no editable fields. The entries
displayed in the following graphic will not be the same as those seen on the
Summary Report for your sample.
BD MultiSET Software
337452 Rev. A
159
Figure 5-5 Summary Report view
NOTICE If there were more entries in the Schedule document, Next appears
instead of Quit. Clicking Next proceeds with the acquisition and analysis of the
next entry on the Schedule document.
8 Click Quit.
If you have not saved the Schedule document or the most recent changes, a dialog
box appears asking if you want to save changes. Clicking More Tests displays the
Samples view, allowing you to continue running more samples.
Using MultiSET Tools and Preferences
BD MultiSET software contains tools and preferences for customizing certain features.
The tools allow you to define reagents and panels. The preferences allow you to change
default settings for BD MultiSET software, such as exporting files and printing reports.
The tools can be accessed from the Tools menu or from buttons that appear on the Test
Prefs view. The preferences can be accessed from the MultiSET menu.
You can practice using the tools in BD MultiSET software by doing the steps described
in the following sections. Refer to the BD MultiSET Software User’s Guide for details
about preferences.
MultiSET Tools
Subset Ranges
Subset Ranges allows you to enter six user-defined reference ranges for each subset
whether it is an absolute count, a percentage, or a ratio.
160
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
1 Choose Tools > Subset Ranges.
2 Enter labels for the User Defined Reference Range you want.
After entering a label, do not press Return on the keyboard, as this will exit the
Subset Ranges dialog. Click in another field to accept the names you just entered.
The following example shows User 1 replaced with Adult and User 2 replaced
with Pediatrics.
3 Choose the population from the Subset pop-up menu.
A BD-defined range is provided along with six user-defined ranges, allowing you
seven different ranges. When you define a reagent panel, you need to select one of
the seven reference ranges that will apply to the panel (see Panel Tools in the
following section). The BD-defined range is the default.
4 Enter the range values for the user-defined Reference Ranges, and click OK.
Panel Tools
Panel Tools allows you to create reagent combinations (panels) that you use regularly.
You can add new panels, remove panels, copy the reagents from one panel to another,
and give the panel a new name.
Nine predefined control panels are provided. Use the control panel to run the
BD TruCOUNT Low, Medium, and High Control beads. You cannot create additional
control panels. Four non–control panels are also provided for analyzing the practice
data files that were installed with BD MultiSET software.
BD MultiSET Software
337452 Rev. A
161
1 Choose Tools > Panel Tools.
The Reagents pop-up menu on the left lists the available reagent types, such as
TriTEST, TriTEST TruC (TruC is short for TruCOUNT), and MultiTEST. The list
below Reagents displays the reagents belonging to the particular Reagent Type.
The Panels pop-up menu lists all the panels that have been defined. The list below
the Panels pop-up menu contains the reagents included in the selected panel.
1 Click Add Panel.
2 Type a unique name, and click Add.
3 Choose a Reagent Type from the pop-up menu.
4 Select a reagent from the Reagents list below Reagent Type.
5 Click Move.
6 Continue adding reagents to your panel until all needed reagents have been
added.
7 Select a reference range from the Reference Range pop-up menu.
8 Click OK to exit Panel Tools.
In addition to adding new panels, you can print panels, edit panels, copy panels,
remove panels, and reorder panels, as described below. Refer to the BD MultiSET
Software User’s Guide for details.
Reagent Tools
Reagent Tools allow you to customize Dot Plot Info, Acquisition Target Info, and FL
Parameter Info for BD-defined reagents.
162
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Reagent Tools also allow you to create user-defined reagents by:
•
Copying a BD-defined reagent and customizing FL Parameter Info, Acquisition
Target Info, Population Colors/Names, Edit Subset Results, and Dot Plot Info.
•
Customizing one of the 15 Attractor Templates provided with MultiSET software
(located in the Templates Reagents folder in the MultiSET folder, in the BD
Applications folder).
•
Customizing an Attractor Set you created in Attractors software.
Customizing Selections for a BD-Defined Reagent
1 Choose Tools > Reagent Tools.
2 Choose a reagent type from the Reagent Type pop-up menu.
3 Choose a reagent from the Reagent pop-up menu.
4 Make selections for the reagent.
Each BD reagent has default (BD-preferred) values. Use these values or modify
them.
•
Select the number of plots you want to view and print for each reagent.
•
Choose the X and Y parameters for each plot by using the pop-up menus.
•
Enter the axis label for each fluorescence parameter.
•
Enter the minimum number of events to acquire for the target population.
•
View a list of subset results produced by selected reagents.
BD MultiSET Software
337452 Rev. A
163
Click Revert to BD Defaults to set all the information for the selected reagent
back to the original BD default values. Refer to the BD MultiSET Software User’s
Guide for more information on BD defaults.
Copying a BD-Defined Reagent
You can make copies of BD-defined reagents and customize selections for the reagent in
Reagent Tools. For example, because analysis of CD3/CD4v4/CD45 reagent
combination is similar to CD3/CD4/CD45 reagent combination, you can copy the BDdefined CD3/CD4/CD45 reagent and make necessary modifications instead of creating
a reagent from scratch.
1 Choose TriTEST from the Reagent Type pop-up menu.
2 Choose the CD3/CD4/CD45 reagent from the Reagent pop-up menu.
3 Click Copy Reagent.
A dialog appears with copy appended to the default name.
4 Enter a unique name or leave the default name, and click OK.
Notice that the Reagent Type has changed to User Defined, and the reagent
selected in the Reagent pop-up menu is the reagent just copied with a UD
appended to the name to indicate it is user-defined. At this point, you can leave
the defaults shown in the copied reagent view or you can make alterations to fit
your analysis needs.
5 When you have completed your selections, click OK in the Reagent Tools view to
save the changes.
Lot IDs
Enter lot IDs for the reagents, controls, and absolute count beads you are using. You
can find this information on the appropriate packaging. You can enter only one lot ID
per Schedule document for each reagent or bead type. Refer to the BD MultiSET
Software User’s Guide for details.
164
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Using User-Defined Reagents
You can create user-defined reagents in the following ways.
•
Copying and then customizing one of the predefined reagents that comes with the
software to analyze BD TriTEST and BD MultiTEST reagent combinations.
•
Using any of the fifteen predefined user-defined templates provided in the
Template Reagents folder in the MultiSET Folder.
To practice using this feature, you can follow instructions provided in the BD MultiSET
Software Tutorial. The tutorial is a PDF file which can be found on your computer hard
drive in the Documentation folder/Tutorials folder. The Documentation folder can also
be found on the BD FACStation Software CD version 5.1 (Part. No. 334978).
Tutorials folder
1 Print the BD MultiSET Software Tutorial located on your computer’s hard drive.
2 Follow instructions provided in the user-defined reagents section.
BD MultiSET Software
337452 Rev. A
165
Using Help
When you need assistance with BD MultiSET software, use the Help viewer to quickly
locate the information.
1 From the Help menu, choose MultiSET Help.
The fastest way to search for help is to use the Ask a Question feature. You can
also search by looking through the table of contents or index.
2 Enter expert lymph gate in the Search field; press Return.
A list of topics appears in the lower portion of the Help view. Double-click on a
topic to go to that section of Help.
3 When you are finished, close the Help viewer by clicking the red close button.
4 Choose MultiSET > Quit MultiSET; click Quit.
166
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
BD MultiSET™
Software
337452 Rev. A
Reagents
BD TriTEST™
BD MultiTEST™
Lyse/No-Wash
Lyse/No-Wash
IgG1/IgG1/45 (CQ)
CD3/CD8/CD45/CD4
CD3/CD4/CD45
CD3/CD16+56/CD45/
CD19
CD3/CD8/CD45
CD3/CD16+56/CD45
2 tubes
CD3/CD19/CD45
4 or 5 tubes
2
1
BD TruCOUNT™
Absolute Count Tubes
• Used with BD TriTEST or BD MultiTEST
reagents to determine absolute counts in
whole blood.
• Contains a lyophilized pellet of a known
number of fluorescent-labeled beads.
3
Process Control
• A “normal” sample prepared and run with
patient samples.
• Controls for antibody staining, red cell lysis,
instrument setup and performance, and
data analysis.
• Sources:
– healthy subject
– BD Multi-Check Control
– BD Multi-Check CD4 Low Control
4
2
BD TruCOUNT™ Controls
• Control for elements of the absolute
counting process such as: pipetting and
BD TruCOUNT Absolute Count Bead
performance.
• Contains vials of Low, Medium, and High
concentrations of beads.
• Provided with expected target value ranges.
• Added to process control sample.
5
Sample Preparation
50 mL whole blood*
*
20µl of TriTEST or20
MultiTEST
mL BDReagent
MultiTEST
+
50µl* of whole blood (EDTA)
BD TruCOUNT™
Absolute Count Tube
reagent
Add to TruCOUNT
Vortex
Absolute Count Tubes
15 Minute Incubation (at room temperature in the dark)
Vortex.
15 minutes
(@ room temperature, dark)
450 mL BD FACSLyse solution (1x)
450µl FACS Lyse
Vortex.
Vortex
15 minutes
15 Minute
Incubation
(@ room temperature,
dark)
(at room temperature in the dark)
50µl* Control Beads (Low, Med, or High)
50 mL BD TruCOUNT Control beads
Acquire and Analyze
Acquire Samples
* Use reverse pipetting technique
6
3
Lyse/No-Wash Optimization
•
Adjust SSC PMT, if
necessary.
•
Raise or lower the
FL3-H threshold level
until the majority of the
debris is removed.
7
1000
Expert Lymphocyte Gating
400
600
400
200
Slice is taken
through lymph
cluster center
and into the
mono cluster
Valley
Monos
0
200
Side Scatter
800
600
Lymphs
Histogram is constructed
from SSC data from slice
0
200
400
600
CD45 - FL3
800
1000
0
100
200
300
400
500
Side Scatter (Gated)
Granulocytes
BD TruCount Absolute Count Beads
Monocytes
Basophils
expert lymph gate
R
8
4
Analysis
BD TruCount Absolute Count Beads
expert lymphocyte gate
9
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Run Lyse/No-Wash assay in BD FACSComp
software.
10
5
Start Up
System
•
•
•
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Place populations on scale in SSC (if needed).
Adjust FL3 threshold to get rid of most of the debris.
Save optimized settings.
TruCOUNT beads
lymphocytes
11
debris
Start Up
System
•
•
•
•
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
Specify data collection information (file name, storage
location, panels, etc.).
Click Acquire.
Gate manually, if needed.
Print or export results, if needed.
12
6
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
13
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
14
7
6
DNA
After completing this module, you will be able to:
DNA
•
Define the parts of the cell cycle.
•
Explain what parameters affect DNA analysis by flow cytometry.
•
Set up and perform QC in preparation for DNA analysis.
•
Acquire DNA data using BD CellQuest™ Pro software.
•
Analyze DNA data using ModFit LT™ software.
337452 Rev. A
167
Overview of DNA
The following section provides an overview of concepts and theory pertaining to DNA
analysis by flow cytometry. The following topics are discussed.
•
Review of the cell cycle
•
Factors affecting DNA analysis by flow cytometry
•
Standardization requirements for DNA flow cytometry
•
BD DNA QC Particles kit
Review of the Cell Cycle
Normal replicating diploid cells pass through four distinct phases: G1, S, G2, and M. All
normal nonreplicating diploid cells are said to be in the resting or G0 phase of the cell
cycle. The DNA is contained in paired chromosomes and the amount is referred to as
2C.
G1 Phase
When cellular reproduction is initiated, cells enter the G1 phase of the cell cycle. RNA
and protein synthesis occur during this phase, but the cellular DNA content remains
constant at 2C. Because G0 and G1 both contain 2C DNA, cells are indistinguishable by
flow cytometric DNA analysis and are noted together as G0/G1.
S Phase
The DNA synthesis, or S phase, begins with replication of DNA molecules. S phase
continues until the entire complement of DNA is replicated, and the DNA content of
the cell is doubled, now referred to as 4C.
G2 Phase
The cell then progresses into the G2 phase of the cell cycle. DNA content is unchanged
as the cell prepares for division, and thus remains at 4C.
M Phase
During this phase the cell undergoes division, or mitosis (M phase). The DNA content
remains at 4C until the cell splits into two daughter cells. Each daughter cell will
possess 2C amount of DNA.
168
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Because G2 and M both contain 4C DNA, cells are indistinguishable by flow cytometric
DNA analysis and noted as G2 + M.
G1
G 1 phase
S
phase
G2 + M
phase
Number of cells
Number of cells
An idealized DNA content distribution is shown in the plot on the left in Figure 6-1.
DNA distributions obtained by flow cytometry will more closely resemble those seen in
the plot on the right in Figure 6-1. G0/G1 and the G2 + M peaks broaden because of
variables such as sample preparation, dye uptake, and instrument factors, as well as
true variation in the DNA content of a cell. Analysis of the DNA distribution allows the
detection of differences in G0/G1DNA content between cell populations, and the relative
proportions of cells in the G0/G1, S phase, and G2 + M phases of the cell cycle.
G +M
2
S
DNA content
1x DNA content
Theoretical Flow DNA Histogram
2x
Observed Flow DNA Histogram
Figure 6-1 Theoretical and observed DNA histograms
DNA Index (DI) and cell cycle percentages can be derived from DNA histograms. DI
(ploidy) is a ratio between the G0/G1 peak channel of the unknown sample and the
G0/G1 peak channel of a standard (eg, human diploid cells, such as lymphocytes). A DI
of 1.0 is said to be diploid. Normal cells containing two copies of each chromosome are
said to be diploid. If the resulting ratio is not equal to 1.0, the sample is defined as being
aneuploid.
Factors Affecting DNA Analysis by Flow Cytometry
Many factors can affect the outcome of your DNA histogram and ultimately your DNA
histogram analysis. See the following list for three possible factors:
Sample Preparation—A single cell suspension representative of the tumor (or sample) in
question is required. Minimal cell/nuclear membrane damage, debris, and aggregates
will improve your DNA analysis.
Staining—Stain with fluorescent dyes that bind to cellular DNA specifically and
stoichiometrically.
Flow cytometer—Acquire data on an instrument that can record the relative DNA
content. Measurements from a histogram should be analyzed by mathematical
algorithms. Information on percent coefficient of variation (%CV) and linearity are
required for the quality control of the data.
DNA
337452 Rev. A
169
Standardization Requirements for DNA Flow Cytometry
Variation in instrument performance or in sample preparation and staining will affect
the data. Therefore, overall system performance must be monitored to ensure stability
and reproducibility, as well as to assist in troubleshooting. A standard can be defined as
a stable particle which has been or could be assigned values for fluorescence or scatter.
Standards typically are used to calibrate instrumentation.
Resolution
Resolution, or precision, is critical in DNA flow cytometry because small differences in
DNA content between cell populations may be biologically significant. The precision is
monitored by measuring the coefficient of variation (CV) of the G0/G1 peak of a DNA
distribution of a standard particle. This is also a measure of the reproducibility of the
system. Percent CV is defined as the standard deviation (SD) divided by the mean times
100:
%CV = (SD/mean) x 100
The resolution of measurements may be affected by instrument factors (for example,
fluidics, alignment, and optics) as well as sample artifacts introduced during sample
preparation and staining. Assessment of true biologic variance in DNA content is of
paramount importance. Minimizing variation due to preparation artifacts and
instrument factors is crucial to optimizing DNA analysis conditions.
Chicken erythrocyte nuclei (CEN) contain approximately 1/3 the DNA content of a
normal mammalian-diploid cell and are not true cycling cells. Therefore, they do not
have an S phase fraction or G2+M fraction. CEN are useful biological particles for
assessing instrument resolution.
Linearity
Assessment of instrument linearity is also important. Linearity is the relationship
between the channel number of a particular population and the intensity of the
measured parameter. In terms of DNA content analysis, this means the peak channel
number of a G2 + M peak should be twice that of the G0/G1 peak. The positioning of the
G0/G1 peaks and the G2 + M peaks provides the benchmark against which other peaks
will be compared. If the instrument is unable to provide a linear response, there will be
no way to gauge whether a population contains an expected normal amount of DNA.
CENs are intentionally formulated in such a way that they aggregate. It is the ratio of
the doublet:singlet peak channel numbers that allows an evaluation of instrument
linearity.
Doublet Discrimination
Another critical area in DNA analysis is doublet discrimination. A doublet is defined as
two particles that pass through the sensing region of the instrument together and are
170
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
recorded as a single event. In terms of DNA analysis, two particles (each possessing 2N
amount of DNA) passing through the sensing region simultaneously are counted as a
single event with a fluorescence intensity of 4N. When this occurs, the number of events
in the 4N channel is increased artificially because of the presence of doublets.
Instruments which are equipped with pulse processing are capable of identifying
doublets for further DNA analysis and elimination.
BD Biosciences recommends the use of calf thymocyte nuclei (CTN) as a control to
validate the ability of the flow cytometer to distinguish doublets from true cycling
G2 + M cells. Since CTN are cycling cells manufactured to form doublets, they are an
ideal biological particle which can be used to monitor this instrument function.
DNA Quality Control Kit
BD DNA QC Particles kit provides a means for testing and documenting optimal
performance of your flow cytometer. The kit is made up of four reagents (chicken
erythrocyte nuclei [CEN], calf thymocyte nuclei [CTN], 2-µm beads, and propidium
iodide [PI]). The CEN are used as a setup tool in conjunction with BD CellQuest Pro
software. They provide information relating to instrument resolution and linearity. The
CTN contain cycling populations of cells which are used to validate performance of the
DDM (Doublet Discrimination Module) or pulse processor, and may also be used as a
control to monitor performance using cell cycle statistics. The kit also includes 2-µm
beads, which are stable particles used to verify instrument alignment.
Test Principles
CEN are prepared by detergent treatment of chicken blood followed by fixation of the
isolated nuclei in ethanol. The CEN preparation is formulated to form singlet, doublets,
triplets, and higher order aggregates. When stained with PI, the CEN form four or more
reference peaks when collected using the FL2-A parameter with BD CellQuest Pro
software. If the instrument response is linear, and the nuclei have been correctly stained,
the channel values of the means of these peaks should be approximately two, three, and
four times the singlet mean channel number.
CTN are prepared by detergent treatment followed by fixation of the isolated nuclei in
formaldehyde. The CTN provide a stable source of nuclei with all phases of the cell
cycle represented. Most of the nuclei are in the G0/G1 phase, and a smaller proportion
of the nuclei are in the S and G2+M phases of the cell cycle.
Two-micron beads are a nonbiological standard used to check instrument alignment
either before or after running the CEN or CTN, especially if the biological particles
should fail to perform as expected. The microspheres function to verify instrument
alignment. They fluoresce in both FL1 and FL2, thereby providing a means of verifying
instrument performance independent of stain or sample preparation.
The PI stain intercalates into the DNA of the CEN and CTN. When excited by blue
laser light (488 nm), the dye will fluoresce in FL2. The intensity of the fluorescence is
proportional to the DNA content of the cell.
DNA
337452 Rev. A
171
DNA Exercises
Acquisition of DNA data on a flow cytometer is a multistep process. The following
figure outlines the main steps involved. In this module, we will focus on instrument QC,
optimization of instrument settings, data acquisition, and data analysis.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
In the following exercises, you will:
•
Perform instrument quality control (QC) using the BD CellQuest Pro DNA QC
document and reagents from the BD DNA QC Particles kit to assess instrument
resolution, linearity, and doublet discrimination.
•
Optimize instrument settings for cells using cytometer controls in
BD CellQuest Pro software.
•
Acquire samples using BD CellQuest Pro software.
•
Perform an automatic and manual analysis of demonstration files using
ModFit LT software.
•
Analyze the samples acquired in class using ModFit LT software.
To perform ploidy and cell cycle analysis on a sample, it is recommended that three
tubes be run for each unknown: a diploid standard, the unknown admixed with the
diploid standard, and the unknown alone. The preferred diploid standard for a tumor
sample is normal tissue of the same tissue type as the tumor. An alternative standard is
peripheral blood mononuclear cells (PBMCs).1
The nuclei from the cells will be isolated and stained with PI using the BD CycleTEST™
Plus kit. In the exercise that follows, you will acquire data from three tubes as described
in the preceding paragraph.
Performing Quality Control
In this exercise, you will perform QC as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Prepare CEN and CTN samples.
• Set up the DNA Experiment document.
• Run CEN particles and determine resolution and linearity.
• Run CTN particles and check doublet discrimination.
172
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
To perform QC, it is necessary to run particles like those found in the BD DNA QC
Particles kit to check the cytometer’s resolution, linearity, and doublet discrimination.
Preparing CEN and CTN Samples
Prepare CEN and CTN samples according to the BD DNA QC Particles kit package
insert. During the training class, samples are prepared for you.
Using the DNA QC Document
The DNA QC document is a BD CellQuest Pro Experiment document provided for you
to check linearity, resolution, and doublet discrimination when used with the BD DNA
QC particles.
Follow the instructions provided in Chapter 2 in the DNA Setup User Guide. Once you
have completed the chapter, return to this page to enter your QC results.
1 Open the DNA Setup User’s Guide.
•
Navigate to the Documentation folder on the hard drive.
If you cannot locate the folder on the hard drive, the folder can also be found
on the BD FACStation Software CD, version 5.1 or later.
•
DNA
Double-click the DNA Setup User’s Guide to open it.
337452 Rev. A
173
2 Print pages 14 - 24.
•
Choose File > Print.
•
Enter the page range in the printer dialog that appears.
•
Click Print.
3 Follow the instructions on the printout to perform instrument QC.
Enter your QC results in the following space provided.
1 What was your instrument’s resolution (%CV of CEN singlet peak)?
_____________________________________________________________________
2 What was your instrument’s linearity (M2/M1 ratio on CEN FL2-A histogram)?
_____________________________________________________________________
3 Could you resolve CTN doublets from singlets? ____________________________
Optimizing Instrument Settings
In this exercise, you will optimize instrument settings, as outlined in the following
graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Optimize FL2 PMT and FL2-W Amp Gain settings.
• Optimize FL2 threshold setting.
As is the case with immunophenotyping, cytometer optimization must be performed
before acquiring DNA data. Next, you will optimize instrument settings for the cells of
interest.
Optimization is the process of assuring that the settings on the cytometer are
appropriate for the samples you are running. In this exercise, the following samples
stained with PI are used.
174
•
PBMCs (peripheral blood mononuclear cells from a normal donor)
•
PBMCs + tumor cells (from a cell line)
•
Tumor cells
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Use the DNA Acquisition document located in the DNA QC folder to optimize FL2
settings. Do the following:
•
Adjust the FL2 PMT voltage to set the PBMC population at channel 200 on an
FL2-area histogram.
•
Adjust the FL2 PMT voltage and FL2-W Amp Gain if needed, to make sure all
cells of interest are on scale and the singlets can be discriminated from the
doublets.
•
Adjust FL2 threshold to exclude most of the debris.
Data should be accumulated over a range of DNA indices from 0.1C to 6C.1 Setting the
diploid peak at channel 200 assures that the data range complies with this
recommendation. Again, FL2 settings are adjusted because cells are stained with PI,
which is detected in FL2.
Optimizing FL2 PMT and FL2-W Amp Gain Settings
1 Choose File > Open in BD CellQuest Pro software.
2 Navigate to DNA QC folder > DNA Acquisition document.
3 Double-click the DNA Acquisition document to open it.
A one-page document appears containing an FL2-W vs FL2-A dot plot. There is
also an FL2-A histogram plot with a corresponding Histogram Statistics box.
4 Verify that the Setup box is checked in the Browser.
5 Verify the flow rate is LO and the cytometer is in RUN.
6 Display Counters, if you want.
Choose Acquire > Counters. It’s always a good idea to display Counters while
you are optimizing and acquiring because a change in event rate might be an
indication that you have bubbles, aggregrates, cells settled out of solution, or you
are running out of cells.
7 Mix and then install the PBMC sample on the SIP; click Acquire.
8 Choose Cytometer > Threshold and verify that the Threshold is set to FL2.
9 Choose Cytometer > Detectors/Amps.
10 While viewing the FL2-A histogram, adjust the FL2 PMT Voltage until the mean
of the PBMC population is approximately at channel 200 ±5.
The channel number can be approximated by viewing the M1 Mean in the
Histogram Statistics view. Click Pause and Restart to refresh the screen, as
needed.
DNA
337452 Rev. A
175
11 Remove the PBMCs, mix and then place the sample containing the admixed cells
on the SIP.
12 While viewing the FL2-W vs FL2-A dot plot, verify that all of the cells of interest
are on scale.
NOTICE With these particular cells, adjustments might not be needed. However,
for some aneuploid samples that are greater than tetraploid, it might be necessary
to adjust the FL2 PMT voltage gain to place all of the populations of interest on
scale for FL2-A.
NOTICE With some samples, it may be necessary to adjust the FL2-W amplifier so
that you can discriminate between G2+M singlets and G0/G1 doublets. In most
cases, the FL2-W amplifier setting that was established during CEN setup is
sufficient.
13 Optimize the FL2 threshold to exclude most of the debris, if needed.
14 Click Pause; then Abort.
15 Install the tumor tube and verify that all events are on scale.
16 Remove the tumor tube and install a tube of DI water.
Acquiring Data
In this exercise, you will acquire data, as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Specify data collection information.
• Display the Counter dialog box, if needed.
• Set the flow rate to LO and deselect the Setup checkbox.
• Click Acquire.
Specifying Data Collection Information
Before you acquire data, you need to specify data collection information such as
number of events to acquire, data file name prefix and suffix, storage location, and so
on.
1 Choose Acquire > Acquisition and Storage.
2 Set the collection criteria to 20,000 for the number of events.
NOTICE When returning to your lab, it is advisable that you set a region around
the singlet events on a FL2-W vs FL2-A plot and set the collection criteria so that
176
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
acquisition will stop when 15,000 gated events are collected. This will ensure that
you have enough singlet events for accurate S phase estimates in ModFit.
3 If necessary, choose Windows > Show Browser.
4 Click the triangle next to Acquisition Controls in the Browser, if needed, to
display the acquisition controls.
5 Choose a storage location for the data files.
During the training class, do the following:
•
Click the Change button to the right of Directory.
•
Navigate to your folder in the student folder.
•
Select your folder; Click Choose.
6 Specify a data file name prefix and suffix.
During the training class, do the following:
•
Click the Change button to the right of File.
•
Verify that the File Name Prefix is set to Sample ID and the File Count is
None.
•
Click OK.
•
Enter the prefix you want in the Sample ID field in the Browser. For example,
PBMC; press Return.
7 Verify that the Setup box is checked in the Browser.
Saving Data Files
1 Remove the admixed sample, mix and then place the PBMC sample back on the
SIP.
2 Deselect the Setup box and click Acquire.
The PBMC data will be stored in the preselected folder. Note the mean channel
and CV of the PBMC. The PBMC or other diploid control is used as a control for
your staining procedure. The CV should be checked daily to track instrument
resolution and to monitor reagent integrity.
3 After the PBMC acquisition is complete, acquire the admixed PBMC + tumor
sample; then acquire the tumor alone sample.
NOTICE Don’t forget to change the Sample ID before acquiring each sample.
DNA
337452 Rev. A
177
4 Save these new DNA instrument settings as a separate file for your records.
•
Choose Cytometer > Instrument Settings.
•
Click Save.
•
Specify your folder in the student folder.
•
Specify a name for the file.
•
Click Save.
•
Click Done.
ModFit LT Software
ModFit LT software is a third-party software created by Verity Software House. The
following section provides an overview of the software. Refer to the ModFit LT
Software User Guide for details.
The software offers two forms of DNA analysis: auto and manual. In this module you
will have the opportunity to exercise both options. ModFit LT software also allows you
to create and store custom report forms, and to output results to a database.
This and other tutorials can also be accessed through the Help menu while using the
program.
Most of the functions in ModFit LT software are accessible through the Toolbar.
178
•
The File button allows you to select and open a data file for analysis.
•
The Auto button instructs the software to perform Auto Analysis on the data.
•
The Scale button allows you to choose what area of the histogram you wish to
model, as well as to adjust vertical scaling of the histogram.
•
The Mod button allows you to input information about the sample and the data
file that helps the software choose an appropriate mathematical model.
•
The Range button allows you to identify areas of the histogram to aid the
software in its analysis.
•
The Fit button instructs the software to perform a manual fit on the data
according to guidelines you have established.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
•
The Diags button opens a window containing statistical information on how well
the software was able to fit the data.
•
The Reprt button places the Report Tools on the page. You use these tools to
construct and edit a report.
•
The Save button automatically names and saves reports.
•
The Print button prints the current report.
•
The Help button gives you access to online help, including tutorials.
ModFit LT Software Files
When using ModFit LT software you will encounter several types of files.
Data Files
These are the files you read into ModFit LT software for analysis. These include both
FCS listmode and histogram files created in BD CellQuest or BD CellQuest Pro
software as well as FCS files created with other software. These data files are accessible
using the File button on the Toolbar, or by selecting File > Open > Data File. Note that
you must specify which type of file you wish to open in order to display it in the
window.
Report Files
ModFit LT software allows you to customize the presentation of your analysis and save
it as a Report File. This allows you to review the analysis at a later date, or to create a
template to standardize future data presentations. The software offers several sample
reports. Reports can be opened by choosing File > Open > Report.
Database Files
You have the option of outputting the results of analyses to a database file. These files
can then be opened by a database or spreadsheet program for further analysis.
Database files can be accessed through the Edit Database option of the Edit menu
(advanced and administrative users only). To open the database automatically when
ModFit LT software is launched, select Edit > Auto-link at startup.
Graphic Files
ModFit LT software has the option of creating a graphic file of the histogram
and/or the report page. These files are saved in a PICT format.
DNA
337452 Rev. A
179
DNA Analysis with ModFit LT Software
In the following exercise, you will analyze DNA data in ModFit LT software, as
described in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Display data.
• Create gates, if needed.
• Perform auto or manual analysis.
• Perform QC of the analysis.
• Create and customize a report, if needed.
• Print or export results, if needed.
In the following exercises, you will:
•
Perform an auto and manual analysis of a demonstration DNA data file.
•
Analyze your acquired data from the previous exercise.
Performing Auto Analysis
In this exercise you will open a data file and perform an Auto analysis of the resultant
DNA histogram.
Displaying Data
1 Launch ModFit LT software from the Apple Menu.
A User Login dialog box appears. See Figure 6-2 on page 180.
Figure 6-2 User Login
The software offers three levels of user groups within the program:
•
180
Standard user
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
•
Advanced user
•
Administrator user
For information on the different permissions for each user group, refer to page 93
of the ModFit LT Software Users Guide.
2 Click in the User Name edit box and enter your name.
3 Choose the User group by selecting Standard User in the pull-down menu.
4 Click OK.
5 Click the File button (
) on the Toolbar.
The Open Data dialog appears.
The software is capable of analyzing several different file types, including
listmode and histogram files created in BD CellQuest or BD CellQuest Pro
software, as well as FCS files created in other software. Only files of the type
specified will be listed in the window.
6 If necessary, click and hold on the pull-down below List Files of Type. Choose All
Files.
7 If necessary, navigate to the BD Applications > ModFit LT folder > Sample folder.
8 Select the folder; then click Open.
9 Select the file Sample1.FCS; then click Open.
The histogram appears on the screen. See Figure 6-3 on page 182. Note that there
are arrowheads under the significant peaks, in this case the G0/G1 and the G2+M
peaks. This means that the software has found them and identified them as
significant.
DNA
337452 Rev. A
181
Figure 6-3 FL2 histogram
Analyzing Data
1 Choose Edit > Automatic Analysis Settings.
The Edit Properties for Auto Analysis settings dialog appears.
2 Make sure both AutoAggregate and AutoDebris are enabled.
We recommend that you enable AutoDebris and AutoAggregates, as these
components are robust and automatic in the way they handle debris and
aggregation in histograms.
182
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
3 Click OK.
The AutoAggregate selection allows the program to model aggregates, whether
they are obviously visible or not.
The Apoptosis option is usually left disabled for Auto Analysis unless you have a
series of histograms in which you want to model apoptosis as a Gaussian
distribution below the first cycle’s G1 peak. There are no apoptotic cells in this
sample.
4 Click the Auto button (
) on the Toolbar.
ModFit LT software begins analyzing the data.
During the analysis, the Modeler Status dialog box appears. This reports on the
progress of the modeling.
During analysis, ModFit LT software attempts to characterize the histogram with
a series of equations. After its initial attempt, the software checks to see how well
its mathematical characterization corresponds with the real data. The difference
between the real data and the mathematical model of the data is indicated by the
RCS, or Reduced Chi-square value. The lower the RCS, the better the fit. After its
initial attempt, the program will adjust its parameters and try again. It will
continue this process for ten iterations, or until the RCS does not decrease
significantly.
When the analysis is completed, the histogram will be displayed and overlaid with
graphical representations of the modeled G0/G1, S-phase, and G2+M populations.
Also displayed are the results of the fit, along with some information about the
data file and the mode of analysis.
Note that the sample was characterized as 100% diploid. Without the use of
internal or external controls, a single population will be reported as diploid.
DNA
337452 Rev. A
183
Other reported results include:
•
Percentages of cells in each phase of the cell cycle
•
Positions of the G0/G1 and G2+M peaks
•
Ratio of the peak positions (an indication of linearity)
•
Percentage of data modeled as debris
•
Percentage of data modeled as aggregates
•
Number of events modeled
•
Cycling events per channel
•
Final RCS
•
Background, aggregates, and debris (BAD)
The Report tools palette is also displayed on the right side of the report. See Creating a
Report on page 185 for details.
Performing QC of the Analysis
The information provided in Fit Diagnostics is designed to indicate how reliable the fit
data is. It compares %CV, number of cells fit, RCS, and percentage of data fit as
background, aggregates, and debris (%BAD) with established guidelines for these
parameters, both numerically and graphically.
1 Click the Diags button (
) on the Toolbar.
The Fit Diagnostics dialog appears. See Figure 6-4 on page 185. The status boxes
next to the category buttons are indicative of how well the fit compares to
guidelines established for each of the categories. Clicking the category buttons
displays additional information on these guidelines.
184
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 6-4 Fit Diagnostics dialog
S-Phase Error analysis reports the error probability distribution of each of the Sphase estimates.
2 Click the %CV button.
You will see additional information regarding guidelines for this category.
3 Click OK when you have read the information.
4 Repeat steps 2 and 3 for the Cell#, RCS, and %B.A.D. buttons.
5 Click OK to close the dialog.
6 Choose File > Print to obtain a hard copy of your results.
Creating a Report
The documents created by ModFit LT software are called reports. Reports contain
analysis information for one data sample, and can be saved to disk and re-opened by
the program.
In this exercise you will use some of the options ModFit LT software provides.
1 Click the Reprt button (
) on the Toolbar if the tools palette is not already
displayed.
DNA
337452 Rev. A
185
The Tools palette appears. These tools assist you in customizing your report.
They can also be accessed individually from the Tool menu.
Table 6-1 Report tools palette description
Adds text to your report.
Displays the Edit Equations dialog box to permit editing of
the analysis equations.
Adds additional fields to your results, including statistics
generated by ModFit LT software and header information
contained in the data file.
Adds lines, rectangles, circles, and arrows to your report.
Adds a graph zoomed on the X-axis to magnify the area near
the diploid G1 component.
Adds a graph object zoomed on the Y-axis to magnify the S
phase and aggregate components.
In addition to using the tools outlined in the preceding table, you can move any
element of the report, such as the histogram or the results box, by clicking and
dragging anywhere in the box. You can resize these boxes by clicking them and
dragging the handles at the corners of the box. Double-clicking a box opens a
dialog box that allows you to change the elements of that box: fonts, tic intervals,
zoom factors, and manual spacing.
Customizing Your Report
1 Change the Zoom pull-down menu to the right of the Toolbar to 75%.
This will give you a better overview of the report.
186
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
2 Click the text tool on the Report tool palette.
The Edit Text dialog appears.
This dialog box allows you to insert text onto a report. In addition, text you may
want to use repeatedly can be stored to disk from this dialog box, and recalled
whenever it is needed.
3 Enter the name of your lab (or make one up).
This will overwrite the text in the dialog.
4 Click the Font button.
A menu appears that allows you to choose the style, size, and color of the font for
this text item.
5 When you have selected a font you like, click OK.
6 Click OK again to leave the Edit Text dialog.
7 Click and drag the box containing the text to an appropriate area on the report.
Click and drag any of the square handles.
If you want to make any changes to the text box, double-click on it. This will
reopen the Edit Text dialog box.
If needed, you can add graphic elements to the report.
When you have created and arranged the elements of the report to your
satisfaction, save the report to disk.
8 Select File > Save.
DNA
337452 Rev. A
187
9 Enter a name for the report, navigate to your folder, and click OK.
Printing the Results
1 Choose File > Print.
2 Click Print.
Performing a Manual Analysis
There are two major steps involved in performing a manual analysis:
•
Setting properties for a manual analysis
•
Setting ranges
Models are dynamically built from a basic set of components based on choices you
make in the Edit Properties for Manual Analysis dialog box. These choices are based on
your visual examination of the histogram. Once your choices are made, the program
then selects the required model components, equations, and ranges. The model is then
named based on its construction, and loaded into the modeling program. Refer to the
ModFit LT Software User Guide for a complete explanation of the components used
and meaning of model names.
After the model is loaded, you may edit it by using either the Quick Model Editor or the
Full Model Editor. You may also save a model to the hard disk and reload at a later
time. This may be especially useful if you have made changes to a model or wish to
archive its construction.
Displaying Data
1 With your report still open, choose 100% from the Zoom command pull-down
menu.
2 Choose File > Open > Data File.
3 Navigate to the Hard drive > Training Files Folder > DNA Practice folder.
4 Select DNAH001 and click Open.
Note that when the histogram appears, ModFit LT software marks only two of
the three prominent peaks with arrow heads. The peak farthest to the left, which
188
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
is the G0 diploid peak, is unmarked. When the software has difficulty locating
peaks on its own, you should perform a manual fit.
To perform a manual fit, you first have to give the software some information
about the sample to help it choose an appropriate model that matches your
assessment of the sample. You also have to set ranges to delineate certain features
of the histogram.
Analyzing Data
1 Click the Mod button (
) on the Toolbar.
This opens a dialog box (see Figure 6-5 on page 190) where you can enter
information about the histogram to be modeled.
Examination of the histogram suggests that there is also a single aneuploid
population which is not tetraploid. There are aggregates (doublets and triplets,
for example) in the sample. The diploid population has no S-phase cells, the
aneuploid population has a distinct G2+M population, and there are no apparent
apoptotic cells.
DNA
337452 Rev. A
189
Figure 6-5 Edit Properties for Manual Analysis dialog
2 Enable both the AutoDebris and AutoAggregates checkboxes.
3 Disable Apoptosis if necessary.
4 For number of cycles, choose 2.
Cycle 2 appears in the left column.
5 For the model template, choose Dip Ane.
The sample we are analyzing contains a diploid control and an aneuploid
population.
6 For the Range positions, choose Compute range positions.
7 Click Cycle 1.
The Edit Properties for Cycle 1 dialog appears.
190
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
8 For G2M, choose disabled.
The sample we are analyzing does not contain a diploid G2+M population.
9 Disable the S-Phase.
The sample we are analyzing contains a diploid control with no S-Phase.
10 Click Cycle 2 and verify that ploidy is Aneuploid, G2M visible peak, and S-Phase
are enabled.
11 Click OK.
Ranges appear that designate significant areas of the histogram. The ranges will
appear overlaid on the histogram.
DNA
337452 Rev. A
191
Which ranges appear depends on the model chosen for the particular histogram.
In this case you will see the following ranges:
•
Debris
•
Dip G1 identifies the diploid G0/G1
•
An1 G1 and An1 G2 which identify the aneuploid G0/G1 and G2+M
respectively
To move the ranges drag on the range label. To adjust the size of the range, drag
on the handle on the right side of the range.
rag here to
.
ove drag
range
here to
Drag here to
size range
drag here to size range
move range
12 If necessary, adjust the ranges.
NOTICE Do not change a range setting unless it is absolutely necessary to do so.
Exceptions to centering the range are for near-diploid and near-tetraploid G0/G1
peaks. These ranges need to be displaced to yield reasonable estimates for the
underlying peaks. When you position a range, the program will graphically
overlay its first estimate of the peak on the histogram.
•
Adjust the Debris range so that the left side identifies the starting point of the
debris. This position should correspond to the channel with the highest debris
counts. The right side position of the debris range is not critical.
•
Place the Dip G1 range over the left most peak in the histogram.
•
If necessary, place the An1 G1 and An1 G2 ranges over the G0G1 and G2M
peaks of the cycling aneuploid population.
Note that there is a small peak approximately twice as far along the x-axis as the
diploid G1 peak. You can rescale the histogram to visualize this more clearly.
13 Rescale the histogram.
Click the Scale button (
) on the Toolbar.
A frame will appear around the histogram with handles at the corners. See
Figure 6-6 on page 193. This frame can be adjusted to designate the portion of
the histogram you wish to model and to zoom in on the Y-Scale. Use the handle in
the upper left to drag the frame down to channel 300 on the Y axis.
192
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 6-6 histogram
The scaling frame can also be used to gate histograms, that is, to select only part
of the histogram for analysis.
NOTICE For this exercise, be sure to keep the left edge of the frame as far left as
possible, otherwise you will not model part of the data.
14 Click the Range button again.
You will see an expanded view of the histogram that will allow you to better
visualize small peaks.
.
15 Click the Scale button again, and drag the frame back up to the top of the display.
16 Click the Fit button (
) on the Toolbar to fit the data.
The Report Tools palette is automatically enabled and appears on the right of the
report.
DNA
337452 Rev. A
193
NOTICE Do not click the Auto button. You are doing a manual fit.
Enter the results of the fit below.
Diploid % ____________________________
Dip %G1 _____________________________
Dip %S_______________________________
Dip %G2 _____________________________
Aneuploid % __________________________
An1 %G1 _____________________________
An1 %S ______________________________
An1 %G2 _____________________________
DI ___________________________________
RCS _________________________________
194
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
17 Click the Y-zoom tool.
A graph object zoomed on the Y-axis to magnify the S phase and aggregate
components is added below the histogram.
Performing QC of the Manual Analysis
Visually inspect your results and compare them to information provided in Fit
Diagnostics. Refer to Performing QC of the Analysis on page 184.
Printing the Results
1 Click File > Print.
2 Click Print.
Analyzing Your Acquired Data
As a final exercise you will use ModFit LT software to analyze the DNA data you
acquired in class.
Verity Software House, Inc. suggests using a four-pass process to analyze your data.
•
DNA
Pass 1—analyze all files using automatic analysis in batch mode and
automatically saving the reports.
337452 Rev. A
195
•
Pass 2—evaluate and review each stored report according to the DNA Analysis
rules from Verity Software House, Inc. Re-analyze data if the rules indicate
change is necessary. If data is re-analyzed, re-save the reports.
•
Pass 3—use batch report processing and run Automatic Linearity Detection on all
reports. Auto Linearity adjustment eliminates analysis variance by fitting the
model with the best linearity setting for G2M and aggregates as possible. Once
the best fit is obtained, the report will automatically be saved.
•
Pass 4—review all reports again to assure agreement with the DNA Analysis
Rules from Verity Software House, Inc.
You will analyze your data using this recommended four-pass process.
Displaying Your Data
1 Navigate to your folder and open the data file containing the PBMCs only.
The Choose Parameter For Analysis dialog box appears.
Since you collected list-mode data in BD CellQuest Pro software, the proper
parameter for the analysis must be selected.
2 Select FL2-A as the DNA parameter; then click OK.
The Define Gates dialog appears.
NOTICE Unlike previous data files, this is a multi-parameter data file, so you have
an option to create a gate. For this exercise, the data will not be gated.
196
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
3 Click OK.
A DNA histogram appears.
If ModFit LT software has successfully located the diploid peak and marked it
with an arrowhead, you can perform an Auto analysis. Notice that there is a
smaller second peak marked with an arrowhead. This peak is most likely made up
of doublets or both doublets and cycling cells.
Analyzing Your Data
1 Choose Edit > Automatic Analysis Settings.
The Edit Properties for Auto Analysis settings dialog appears.
2 Make sure that both AutoAggregate and AutoDebris are enabled; then click OK.
We recommend that you enable AutoDebris and AutoAggregates, as these
components are robust and automatic in the way they handle debris and
aggregation in histograms.
DNA
337452 Rev. A
197
3 Perform an automatic fit on the data by clicking the Auto button on the Toolbar.
The fitted histogram and statistics appears.
4 Note the mean channel number of the diploid G1.
Enter this number below. You can refer to this when performing the analysis of
the admixed population to assure that the diploid peak has been properly
identified.
PBMCs Diploid G1 mean channel number: _________________________________
Performing Batch Data Analysis—Pass 1
ModFit LT software offers batch analysis control to provide both data file and report
processing. In this exercise, you will:
•
Batch analyze the data files you just acquired and automatically save the reports.
•
Evaluate and review each report.
•
Use batch report processing and run AutoLinearity on all reports.
•
Do a final review of all reports.
1 Choose Open; then choose File > Open > Data File.
The Open Data File dialog appears.
2 Navigate to your folder and select the three files you acquired.
To select all files, hold down the Shift key as you click each.
198
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
3 Click Open.
When more than one file is selected, the File Batch Toolbar appears at the lowerleft corner of the screen. The Parameter for Analysis dialog appears.
4 Select FL2-A as the DNA parameter; then click OK.
The Define Gate dialog appears.
5 Click OK.
The PBMC DNA histogram appears.
6 Choose Edit > File Batch Settings.
The Edit Properties for File processor dialog appears.
7 Choose the following Current Values.
•
Analysis: choose Auto Analyze.
•
Save report: enable the Save Report option.
•
Save report path: click folder and navigate to your folder.
•
Print report: enable the Print Report option.
•
Save graphics: choose Histogram.
•
Database prompt: choose Disable.
•
File prompt: choose Disable.
8 Click OK.
DNA
337452 Rev. A
199
9 Click the redo button (
) on the file batch Toolbar.
Even though we have already analyzed the first file, reanalyzing will allow the
report to be saved automatically.
10 Click the Auto-Advance button (
) on the Toolbar.
The next files are automatically analyzed and printed. In addition, the reports and
the PICT files of the histograms are saved.
11 Navigate to your student folder and view the contents.
Evaluating and Reviewing Each Report—Pass 2
1 Choose Edit > Report Batch Settings from the Edit menu.
Alternatively, click the Data Files Batch Properties button (
Toolbar.
) on the file Batch
The Edit Batch Properties for Report Processor dialog appears.
2 Choose the following.
•
Analysis: No analysis.
Choosing No analysis will not repeat the previous automatic analysis.
200
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
•
Save report: Don’t re-save report.
Not resaving the report will prevent any accidental overwriting of a report.
•
Print report: Disable (deselect checkbox).
•
Save graphics: No.
•
Database prompt: Disable (deselect checkbox).
3 Click OK.
4 Choose File > Report > Open.
5 Select your three data files and then click Open.
The File Batch Toolbar disappears. The Report Batch Toolbar appears at the
lower-right corner of the window.
The first report is automatically loaded.
6 Review the report.
7 Click the Next Batch item button (
) on the Report Batch Toolbar.
This allows you to scroll through the reports one at a time and do a final review.
NOTICE If any changes are made, be sure to manually save the report as it will not
be saved automatically.
Detecting Linearity Automatically–Pass 3
A good idea after performing automatic analysis is to evaluate and review each stored
analysis report and then run an automatic linearity adjustment on all of the saved
reports.
1 Choose Edit > Report Batch Settings.
The Edit Properties for Report Processor dialog appears.
DNA
337452 Rev. A
201
2 Select the following.
•
Analysis: Fit and do autolinearity.
This allows the current report model to be used and then will apply the
AutoLinearity function to determine the best fit of the data. After the best fit
is obtained, the report will be automatically saved.
•
AutoAddZoomObjects: Enable.
•
Save report: Re-save report.
•
Print report: Enable.
•
Save graphics: No.
•
Database prompt: Disable (deselect checkbox).
3 Click OK.
4 Click the Auto-Advance button (
) on the report Batch Toolbar.
ModFit LT software performs the AutoLinearity adjustment on the entire batch.
Reviewing Reports—Pass 4
After AutoLinearity is complete, it is appropriate to do a final review of the optimized
reports to be sure that underlying model assumptions are still valid.
1 Choose File > Open > Report.
2 Select all three reports and then click Open.
3 Click the Get Next Batch item button (
) to scroll through the reports one at a
time and do a final review.
202
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Creating a Database with ModFit LT Software
ModFit LT software has an easy, yet robust, database system that allows you to store
large amounts of data easily in one or more database files. The databases are created in
a simple text format known as tab-delimited ASCII text, which can be read by common
database and spreadsheet programs. You can save any statistic it generates, as well as
keyword information, to the database.
NOTICE In order to do this exercise, you must change the user log-in to advanced or
administrator user. Standard users may use the database to store results, but only
advanced and administrative users have privileges to change or create a database.
Setting Up the Software
1 Choose File > Change User Login.
2 Click in the User Name field and enter your name.
3 Choose the User group by selecting Advanced User in the pull-down menu.
4 Click in the Password field and type Epsilon as the password.
5 Click OK.
Before you set up your database, you should analyze a file so you can choose all
the statistical keywords associated with the analysis. You should auto-analyze an
aneuploid file to get all of the statistics into the database.
6 Click the File button on the Toolbar.
7 If necessary, navigate to the BD Applications folder > ModFit LT folder > Samples
folder.
8 Select Sample 2.FCS; then click Open.
9 Click the Auto button on the Toolbar.
10 Choose Edit > Edit Databases.
DNA
337452 Rev. A
203
The Edit All databases dialog appears.
You can select or create a database as well as edit, copy, or delete a database.
11 Click Database 1 to select it.
12 Click Copy.
Database 2 appears with the name field highlighted.
13 Click in the Database 2 name field and type TRAINING.
This will be the name of your database while you are in class.
14 Click Edit.
204
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
The Edit Keyword Map dialog appears.
The left side of the dialog displays the Keyword Groups and the Keywords list. As
you choose keywords, they will be displayed on the right side of the dialog.
15 Click Analysis keywords under Keyword Groups.
The keywords belonging to the Analysis keyword group are displayed in the
Keywords list.
16 Highlight each of the following keywords one at a time; then click Add.
DNA
•
Z_AN1_DI (The DI of the aneuploid population.)
•
Z_AN1_G1_MEAN (The mean channel number of the aneuploid G0/G1
peak.)
•
Z_AN1_G2_MEAN (The mean channel number of the aneuploid G2/M
peak.)
337452 Rev. A
205
•
Z_AN1_S (The S phase percent of the first aneuploid.)
•
Z_DIP_G1 (The percentage of diploid cells in G0/G1.)
•
Z_DIP_G1_MEAN (The mean channel number of the diploid G0/G1 peak.)
•
Z_DIP_G2 (The percentage of diploid cells in G2/M.)
•
Z_DIP_G2_MEAN (The mean channel number of the diploid G2/M peak.)
•
Z_DIP_S (The percentage of diploid cells in S phase.)
NOTICE Each keyword you add appears in the Keyword-Database Map list. If
you make a mistake, you can click on a keyword in the Keyword-Database Map
to highlight it; then click Delete to remove it from the list.
Now the database contains the results keywords.
17 Click the Results folder under Keyword Groups.
18 Click the File Keywords folder under the Keywords list.
206
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
19 Click Add.
When the entire folder is selected, all of the file keywords appear in the KeywordDatabase Map.
20 Click OK.
The Edit All databases dialog reappears.
21 Click the Properties button.
The Edit Properties for Training dialog appears.
22 Enable each of the following.
DNA
•
Active—stores records of information in the database.
•
Prompting—allows a message to appear each time ModFit LT software is
about to save a record to the database.
337452 Rev. A
207
•
Viewer—displays the Database Viewer window while you work.
•
Viewer—font size: 12.
NOTICE The default storage location of the database you just created is the BD
Applications folder > ModFit 3.1 folder. The name of the database file is
Training.txt. This is the tab-delimited ASCII text file that you can open in a
spreadsheet application such as Microsoft® Excel.
23 Click OK to close the Edit Properties for Training dialog.
The Database Viewer appears.
24 Close the database viewer.
Now you will create three entries into the database called TRAINING.
Generating the Database
1 Click the File button on the Toolbar and choose Sample1.FCS.
2 Click Open.
3 Perform an auto analysis.
A dialog appears.
208
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 Click Yes.
The Database Viewer for TRAINING appears. As you analyze the remaining files,
the contents of the database are displayed.
5 Analyze Sample2.FCS and Sample3.FCS.
You should now have three entries in the viewer.
6 Choose File > Quit.
Don’t save the document. This closes Modfit LT software.
DNA
337452 Rev. A
209
Viewing the Results in Microsoft Excel
1 Choose Microsoft Excel in the Finder.
2 Select Excel Workbook; click OK.
3 Choose File > Open.
4 Navigate to your database file.
5 Click on the Training.txt, and then click Open.
The Text Import Wizard appears.
6 Make sure the radio button next to Delimited is selected and click Next.
7 Make sure the radio button next to Tab is selected and click Next.
8 Click Finish.
9 Choose File > Print. Click Print.
10 Choose File > Quit.
Setting S-Phase Cutoffs
ModFit LT software has a feature that allows you to create S-phase cutoffs for samples
from your own laboratory. You can categorize your % S-phases as low, intermediate, or
high and have an S-phase assessment displayed on the report. You also have the option
of reviewing and editing the S-phase cutoffs you create.
NOTICE In order to set S phase cutoffs, you must log in to ModFit LT software as an
Administrator. Additionally, you must enter your own cutoff values. Do not use the
default values.
In the following exercise, you will created cutoffs for a kidney tissue and use the cutoff
values entered to perform analysis on a sample file.
1 Choose File > Change User Login.
A User login dialog appears.
2 Choose the User group by selecting Administrator User in the pull-down menu.
3 Enter Omega as the Password.
The passwords for Advanced and Administrator users are located inside the
ModFit LT software box.
210
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 Click OK.
5 Click the File button on the Toolbar.
The Open File dialog appears.
6 Navigate to the BD Applications folder > ModFit LT 3.1 folder > Samples folder.
7 Select Sample1.FCS; then click Open.
8 Choose Edit > S-Phase Cutoffs.
The S-Phase Cutoffs dialog appears.
Figure 6-7 S-Phase Cutoffs dialog
9 Click the box next to Activate Assessment System to select it.
When you check Activate Assessment System in this dialog box, ModFit LT
software will categorize the S-phase percentage of each population analyzed in a
histogram as Low, Intermediate, or High, depending on the percentage of the
population in S phase.
In Figure 6-7 the current tissue type is breast, and the example cutoff points for
diploid cells are 5.0% for Low/Intermediate and 10.0% for Intermediate/High.
This means that if a diploid population in this sample has under 5% of its cells in
S phase, it will be reported as having a low diploid S phase; if it has between 5%
and 10% in S phase it will be reported as having intermediate diploid S, and if it
has over 10% it will be characterized as having a high diploid S phase.
Note that there are also cutoff values for aneuploid populations of each tissue
type, and for percentage of S phase cells for the sample as a whole.
NOTICE ModFit LT software has example cutoff points for different tissue types.
These cannot be used until appropriate values have been established in your lab.
Those that have been edited are designated with an asterisk.
DNA
337452 Rev. A
211
Turning on the S phase assessment feature also causes the program to estimate a
probability distribution about its S phase estimates. This information is available
by clicking the Diag button on the Toolbar.
10 Double-click (or click and drag) on the word in the box under Tissue to highlight
it.
NOTICE In the following graphic, the default tissue type is All. It might be
different on your computer.
11 Enter Kidney.
12 Change the Aneuploid Lo/Int cutoff to 5.0% and the Int/Hi cutoff to 15%.
13 Click the Add button at the lower-left corner of the dialog.
Kidney now appears on the tissue list with an asterisk next to it. See Figure 6-8 on
page 212.
Figure 6-8 Kidney value edited
Note, however, that it is not the current tissue. You can make it the current tissue
in two ways: either by double-clicking it in the tissue list in this dialog, or by
selecting it from the tissue type pull-down located to the right of the Toolbar.
14 Click OK to close the S-Phase Cutoffs dialog box.
15 Click and hold the pull-down tissue type pull-down menu to the right of the
Toolbar; select Kidney.
212
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Analyzing Data with S-Phase Cutoffs Enabled
1 Choose File > Open > Data File.
This is an alternative to opening a file using the File button on the Toolbar.
The Open Data dialog appears.
2 If necessary, click and hold the pull-down menu next to List Files of Type and
choose All Files.
3 Navigate to the BD Applications folder > ModFit LT 3.1 folder > Samples folder.
4 Select SAMPLE1.FCS; click Open.
Note that the software finds both significant peaks in the histogram.
5 Perform an Auto fit on the data.
After the program has finished its last iteration, the Model Status displays the
message Creating S phase probability distribution.
The S phase assessment results are displayed under the histogram. According to
the diploid values assigned to kidney tissue, the S phase of this sample is high.
The calculated p value is an estimate of the probability that this classification is
correct. In this case, the probability is over 99%.
6 Perform QC of the analysis.
7 Print and review the results.
DNA
337452 Rev. A
213
8 Enter the results of the fit in the spaces provided.
% G1 ________________________________
% S phase ___________________________
% G2+M_____________________________
RCS ________________________________
Multiparameter Gating Using ModFit LT Software
A listmode file can have zero, one, or two active gates. If two gates are active, they are
treated like Gate 1 AND Gate 2, where an event must satisfy both gates to be displayed
in a histogram. Recall that AND is a logical operator, similar to operators in
BD CellQuest Pro software.
In this exercise you will be using two gates to process the data file. One gate will be set
around the singlets using an FL2-W vs FL2-A dot plot. The second gate will be set
around FITC-positive events.
In this exercise you will analyze cells that were stained for both DNA and antigen sites.
A fine-needle biopsy of a colon carcinoma was split into three samples.
The file COLON001 represents cells stained with a isotype control and will be used to
set a gate on FL1 fluorescence.
COLON002 was stained with CD45, a pan-leukocyte marker. The FL1 positive
peripheral blood leukocytes in this file will be used to determine the diploid channel
position.
COLON003 was stained with CAM5.2, a marker for cytokeratin. This will stain only
the tumor cells and any normal epithelial tissue included in the sample. By gating on
this marker, we can be assured that we are analyzing the correct population. This also
provides the best diploid control: normal tissue of the same type as the tumor.
Displaying Data
1 Choose File > Open > Data File.
2 Navigate to the Training Files folder > DNA Files folder.
3 Select COLON001; then click Open.
The Choose Parameter dialog appears.
214
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 Select FL2-A as the parameter for DNA; then click OK.
The Define Gates dialog appears.
You will set two gates on this data. The first will be a gate to exclude aggregates.
5 Click the Define Gate 1 button.
6 Select P5: FL2-W as the X parameter and P4: FL2-A as the Y parameter; then
click OK.
The Define Gating Region dialog appears.
7 If necessary, click Enhance small pops.
This will enable you to better visualize the minor populations by maximizing
color differences.
8 Click four times on the + box.
This will further clarify the minor populations.
DNA
337452 Rev. A
215
Gating Data
1 Click the Rectangle button.
2 Set a gate that will exclude aggregates.
Do not exclude the low level debris, as you will want to subject this to modeling.
Likewise, be sure to extend your gate to the top of the plot so as not to exclude
any significant aneuploid populations.
3 When you are satisfied with your gate, click OK.
The second gate you set will be used to select cells that are positive for the
fluoresceinated markers.
4 Click the Define Gate 2 button n the Define Gates dialog.
The Define Gate Parameters dialog appears.
216
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
5 Select P4: FL2-A as the X axis parameter and P3: FL1-H as the Y axis parameter;
click OK.
The Define Gating Region dialog appears.
6 Activate the Enhance small pops feature and click the + button four times to
better visualize the minor populations.
7 Click the Rectangle button.
8 Drag the vertices to set a gate.
It is important to draw the gate so that it conforms closely with the upper edge of
the data. The cells you’ll be looking for in the next data file will not be brightly
stained.
9 When you are satisfied with your gate, click OK.
DNA
337452 Rev. A
217
10 Click OK in the Define Gates dialog.
A histogram with very few cells will appear. This is to be expected, since there are
no FL1-positive cells in this file. Remember, we were only using this file to create
the gates.
Locating the Diploid Channel
Now you will open the COLON002 file. Using the gates you just created you will find
out what channel the CD45 positive cells fall in. This will tell you where you can expect
to find diploid cells.
1 Open the file COLON002. Click OK in the Choose DNA parameter and Define
Gates dialogs.
You should see a peak at about channel 55.
218
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
If you do not see this, try resetting region 2 closer to the data. You will have to
reopen COLON001 to do this.
2 Perform either an Auto or manual analysis on the data.
In all likelihood the fit was not a good one, since there is so little data in the
histogram. Remember that you are using this data file to locate the diploid peak
position. Note the diploid peak position.
Diploid Peak Channel Number: __________________________________________
Analyzing the Tumor Sample
1 Open the file COLON003.
2 Click OK in the Choose DNA parameter dialog.
3 Click the Define Gate 2 button in the Define Gates dialog.
The gate 2 parameters should be set as they were previously.
4 Click OK.
5 Perform a manual fit on the data.
Report the results in the spaces provided.
COLON003 Manual Analysis
Diploid % ____________________________
Dip %G1 _____________________________
Dip %S_______________________________
Dip %G2 _____________________________
Aneuploid % __________________________
An1 %G1 _____________________________
An1 %S ______________________________
An1 %G2 _____________________________
DI ___________________________________
RCS _________________________________
DNA
337452 Rev. A
219
References
1 Shankey TV, Rabinovitch PS, Bagwell B, et al. Guidelines for Implementation of
Clinical DNA Cytometry. Cytometry. 1993; page 474.
Refer to the Flow Cytometry Resource Booklet for additional references.
220
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
DNA Cell Cycle
Analysis
337452 Rev. A
Mitotic Cycle of a Cell
Mit o sis
M
.
G2
Po st - D N A
Sy n t h e s i s
4 hrs
1 hr
G0
G1
Pr e - D N A
Sy n t h e s i s
S
9 hrs
10 hrs
DNA
Sy n t h e s i s
2
1
Mit o sis
M
.
G2
4 hrs
1 hr
G0
G1
Pr e - D N A
Sy n t h e s i s
S
9 hrs
DNA Content
Po st - D N A
Sy n t h e s i s
4C
o
2C oooooooo o
o
oo
o
oo
o
oo
o ooooo o
ooo
10 hrs
DNA
Sy n t h e s i s
S
G2 M G1
Cell Cycle Phase
G1
Cell Cycle Phases
DNA Mass vs. DNA Content
3
4C
o
oo
oo
o
o
oo
oo
2C ooooooooo
G1
S
oooooo
ooo
G2 M G1
Number of Cells
.
G1 phase
G2 + M
phase
S
phase
1x
2x
Cell Cycle Phase
DNA Cont ent
DNA Mass vs. Time
Theoretical Flow DNA Histogram
4
2
G1 phase
G2 + M
phase
S
phase
1x
Number of Cells
Number of Cells
G1
G2 + M
S
2x
1x
2x
DNA Content
DNA Content
Theoretical Flow DNA Histogram
Observed Flow DNA Histogram
5
Flow Cytometric DNA
Measurements
• Cell Cycle Phase Determination
– Software Modeling
– Multivariate Studies: Immunoploidy
• DNA Index
6
3
Flow Cytometric DNA
Measurements
Counts Full Scale
Cell Cycle Phase Determination
Channel Number
7
Flow Cytometric DNA
Measurements
BrDU FITC
.
G0 / G1
G2+M
Propidum Iodide
8
4
Flow Cytometric DNA
Measurements
• DNA Index (DI): Ratio of DNA Content of
Specimen vs. Control
G0/G1 DNA content of Specimen Cells
G0/G1 DNA content of Diploid Standard Cells
9
Flow Cytometric DNA
Measurements
10
5
Flow Cytometric DNA
Measurements
• DNA Index: Expression of DNA Ploidy
DI
Ploidy
Diploid
Haploid
Tetraploid
Aneuploid
Hyperdiploid
Hypodiploid
1.0
0.5
2.0
≠ 1.0
> 1.0
< 1.0
11
Instrument Quality Control for
DNA
Start Up
Cytometer
•
•
•
•
Perform
QC
Optimize
Settings
Record
Data
Analyze
Data
Shut Down
Cytometer
Instrument Setup
Resolution
Linearity
Doublet Discrimination
12
6
Resolution
• Coefficient of Variation (CV) is used to
assess resolution
• CV is defined as the standard deviation
divided by the mean
• The lower the CV, the “tighter” the data,
and the greater the precision
13
Resolution
Poor Resolution (CV ~10%)
Two Peaks
Good Resolution (CV ~3%)
Three Peaks
14
7
Resolution
Better resolution (less peak spread, smaller
CVs) results in:
• A better chance that different populations of similar
DNA content can be seen as two distinct DNA
peaks.
• More accurate mathematical modeling
15
Resolution
Factors affecting resolution:
•
•
•
•
Instrument Alignment
Instrument Cleanliness
Sample Preparation
Flow Rate
16
8
Linearity
Linearity is an expression of how accurately
a population’s channel position correlates
with its DNA content
17
.
Linearity
inst rument t hat is
G2 + M cells
at t wice t he
number of G0 / G1
G2/G1 = 2.0
532
Counts Full Scale
On an
linear,
appear
channel
c e lls.
0
200
400
FL2-A
600
800
18
9
Linearity
An instrument must be linear in order to:
• Accurately report DNA indices
• Perform analysis on complex histograms
19
Linearity
532
0
200
400
FL2-A
600
800
20
10
Linearity
Factors Affecting Linearity:
•
•
•
•
Instrument Alignment
Laser beam size
Laser beam shape
Pulse analysis mode
21
Pulse Height
Volts
Pulse Measurements
Pulse Area
0
Pulse
Width
Time
(µ Seconds)
22
11
Linearity
Analysis of Pulse Area rather than Pulse
Height enhances linearity.
Doublet/Singlet
2.1
.
2.0
FL2 -A Rat io
FL2 -H Rat io
1.9
1.8
0
2
4
6
8
10
12
Bead Diameter (microns)
23
Doublet Discrimination
Importance:
G0/G1 artifactual doublets have the same
DNA content as G2+M cells.
Unless you can distinguish between them,
you will have falsely elevated G2+M
percentages.
24
12
Doublet Discrimination
G1
FL 2
Doublet
G2 /M
Ce ll
FL 2
Time
Time
25
Doublet Discrimination
G1 doublets can be discriminated from
G2+M cells in two ways:
• Pulse height analysis
• Pulse width analysis
26
13
Doublet Discrimination
G0/G1
doublets
G0/G1
G2/M
doublets
G2/M
G0/G1
S
S
G0/G1
27
14
DNA QC
337452 Rev. A
Verification of Instrument
Performance and Quality Control for
DNA Analysis
• BD DNA QC Particles Kit (Cat. # 349523)
• Kit Components
–
–
–
–
–
Kit insert
Vial A: Chicken Erythrocyte Nuclei (CEN)
Vial B: Calf Thymocyte Nuclei (CTN)
Vial C: 2-µm Fluorescent Beads
Vial D: Propidium Iodide (PI)
2
1
Big Picture
Start Up
Cytometer
Perform
QC
Optimize
Settings
Record
Data
Analyze
Data
Shut Down
Cytometer
• Prepare CEN and CTN samples
• Set up the DNA Experiment document
• Run CEN and determine resolution and
linearity
• Run CTN and determine doublet
discrimination
3
Prepare CEN and CTN Samples
1000 uL Propidium Iodide
40 uL CEN
40 uL CTN
Gently Vortex
10 Minute Incubation (at room temperature in the dark)
Perform QC
4
2
Set Up the DNA QC Document
DNA folder
document
5
DNA Experiment Document
6
3
Determine Resolution and
Linearity with CEN
• Adjust FL2 PMT settings to place CEN singlets at channel
200 + 5
2. Adjust FL2-W Amp Gain
1. Adjust FL2 PMT Voltage
7
Determine Resolution and
Linearity with CEN
• Determine resolution (CV of M1 peak) and calculate linearity
(M2/M1)
M1 CV ≤ 3.00
Linearity range = M2/M1 = 1.95 to 2.05
8
4
Determine Doublet
Discrimination with CTN
G2/M singlets
G0/G1 singlets
G0/G1 doublets
9
5
DNA Optimization and
Acquisition
337452 Rev. A
Big Picture
System
Start Up
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
System
Shut Down
• Prepare samples
• Set up the DNA acquisition document
• Optimize FL2 PMT and FL2-W amp gain
settings
• Optimize FL2 threshold setting.
2
1
Set up the DNA Acquisition
Document
DNA folder
document
3
DNA Optimization and Acquisition
Document
4
2
Samples for Optimization,
Acquisition, and Analysis
• PBMCs (peripheral blood mononuclear
cells from a normal donor)
• PBMCs + Tumor cells (from a cell line)
• Tumor cells
5
Optimization
Using the PBMC tube, adjust FL2 PMT voltage to place PBMC
singlets at channel 200 + 5.
6
3
Optimization
Using the PBMCs + Tumor cells tube, make sure all
significant populations are on scale.
7
Optimization
Using the Tumor cells tube, Make sure all significant
populations are on scale.
8
4
Big Picture
System
Start Up
•
•
•
•
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
System
Shut Down
Specify data collection information
Set the flow rate to LO
Deselect the Setup checkbox
Click Acquire
9
5
7
Sorting
After completing this module, you will be able to:
Sorting
•
Explain the basic sorting mechanism.
•
Perform a sort.
•
Verify sort purity.
•
Maintain the sort lines.
337452 Rev. A
221
Sorting Overview
The flow cytometer uses a flow cell system where the cells travel from the bottom of the
flow cell to the top. Positioned slightly above the viewing orifice inside the flow cell is a
catcher tube, which can collect selected cells. As a cell passes through the laser, the
cytometer electronics quickly decide if it is a cell of interest (target cell) based on the
sort gate criteria. Once this is established, the decision to capture a target cell is
determined by the sort mode you select prior to sorting. When the decision is made to
capture the target cell, the electronics wait a fixed period of time to allow the cell to
reach the catcher tube before triggering the catcher tube to swing into the sample
stream to capture the cell. The maximum possible capture rate of the catcher tube is
300 movements/sec. When there is no cell to sort, the catcher tube remains in a resting
position in the sheath stream. Figure 7-1 illustrates the catcher tube in the resting
position and in the cell-capture position. Because the catcher tube is positioned in the
sheath stream while it waits for a target cell, it continuously collects sheath fluid in
addition to the sorted cells, resulting in a dilute sample. After sorting, the collection
tubes must be centrifuged to concentrate the cells.
sorted cells
waste
Catcher
Tube
catcher tube
sheath
Figure 7-1 Cell non-capture or resting position (graphic on the left) and cell capture position (graphic on the
right)
Collection Station
The collection station is where the sorted cells are collected into 50-mL tubes. Tubes
can be placed into three collection ports in the collection station (Figure 7-2 on
page 223). The instrument detects how many tubes have been installed and fills each
tube starting with the left most (tube 1). It takes approximately 9 minutes to fill each
tube with 40 to 45 mL of fluid. If the collection tubes fill before any of the end sorting
criteria has been met, sorting continues but the sorted sample is sent to the waste
222
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
reservoir. If you wish to continue sorting after the collection tubes are filled, replace the
collection tubes with clean BSA-coated tubes, click Pause, then Restart or Resume.
1
2
3
Figure 7-2 Collection tubes
Sort Line Purge Button
Your instrument may or may not have the sort-line purge button, depending on the
options you purchased. When equipped with a purge button, pushing the button
automatically rinses the sort line. The purge button can be used to check if the sort lines
are clear before beginning a sort, and for clearing the sort lines after a sort.
purge button
Sorting
337452 Rev. A
223
Sort Modes
The sort envelope is the area of the sample stream that the catcher tube collects as it
captures a target cell. The size of the envelope reflects the amount of time the catcher
tube remains in the sample stream to capture the cell. Although this envelope contains
the target cell, it may also contain a non-target cell, thus creating a conflict. Should the
catcher tube sort a cell if a non-target cell will be sorted along with it? That is where the
sort mode comes into play. The sort mode determines whether or not to sort a cell when
a conflict occurs. The following figure provides examples of how the catcher tube
decides to sort a cell for each sort mode.
Recovery
Recovery
The selection of the sort mode is made according to the percent of target cells, as well as
the objectives you wish to achieve with the collected cells. For example, if you are
sorting a rare population, you may be willing to obtain lower purity to sort the
maximum possible number of target cells.
The available sort modes are:
224
•
Single Cell–A sort occurs whenever a single target cell is identified in the envelope.
If any additional cell is located within the sort envelope, the envelope will not be
sorted. The result is high purity with less emphasis on recovery. Single- cell mode
also gives you increased count accuracy. For example, if 100 cells is entered in the
Sort Count field, the instrument will sort 100 single cells.
•
Recovery–A sort occurs whenever an envelope is identified as having a target cell,
even if a non-target cell is also in the envelope. Additionally, if another target cell
is located just outside the envelope, the catcher tube stays in the stream for a
longer period of time to capture it as well. The result is a capture of as many
target cells as possible (high recovery) with less emphasis on purity.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
•
Exclusion–A sort occurs when a target cell is identified and there are no nontarget cells in the sort envelope. Also, if a second target cell is located just outside
the sort envelope, no special attempt is made to capture this additional target cell.
The result is high purity with recovery that falls between Single Cell and
Recovery.
Sample Concentration and Selection of the Sort Mode
* Multiply by 12 to get cells sorted
(cells/mL)
Figure 7-3 Sort Rate at Various Event Rates and Target Cell Percentages
The maximum sort rate occurs when the event rate is 2000 events/sec. A sample
concentration of 107 cells/mL will achieve this event rate at LO flow rate. If the
concentration increases, the event rate increases. If the concentration decreases, the
event rate decreases. If the event rate falls above or below 2000 events/sec, the sort rate
decreases.
When an input concentration of 107 cells/mL is used, the sample concentration may
need to be readjusted to achieve an event rate of 2000 events/sec. This can happen
because the LO flow rate may not be exactly 12 µL/min, (it is specified as 12 ±2 µL/
min), or because a hemocytometer count rarely accounts for the red cells, platelets, and
debris that the cytometer detects.
If you are starting with a particularly low number of cells, it may be best to use an event
rate <2000 events/sec to decrease coincidence and maximize recovery in Single Cell
mode.
Sorting
337452 Rev. A
225
Unlike the Single Cell and Exclusion modes, the sort rate in the Recovery mode will
continue to increase as the total rate increases above 2000 events/sec (up to at least
25,000 events/sec), provided the 300 sorts/sec rate has not been exceeded. Since
coincidence is ignored in the Recovery mode, the percent purity will decrease as the
total sample rate increases, due to the increasing probability of non-target cells in the
sort envelope.
Use Recovery mode when recovery of target cells is the primary concern. Purity will go
down as the event rate goes up. This occurs because the likelihood of a coincident nontarget cell in the sort envelope increases with the event rate. The system should continue
to sort at event rates well over the 2,000 events/sec rate that is optimum for Single Cell
mode.
Effect of Event Rate on Sort Rate
It is important to understand the relationship between the event rate and the sort rate.
Figure 7-3 on page 225 illustrates this relationship when the sort mode is in Single Cell
mode. Notice that the maximum sort rate for any given percentage of target cells occurs
at an event rate of approximately 2000 cells/sec. For example, at an event rate of 2000
cells/sec and a target percentage of cells at 60%, the sort rate is 300 cells/sec. This is the
maximum of all sorting rates.
NOTICE If your target population percentage is below 30%, you cannot reach the 300
sorts/sec maximum sorting rate in Single Cell mode. For instance, a 10% target
population sorted at 2000 events/sec should give a sort rate of 50 to 100 sorts/sec.
Sort Windows
The following section describes the different windows in CellQuest that will be used
when performing a sort.
Sort Setup
Sort Setup is selected from Acquire menu to display the Sort Setup window. See
Figure 7-4 on page 227. The selection of the sort mode is made according to the percent
of target cells, as well as the objectives that you wish to achieve with the collected cells.
For example, if you are sorting a rare population, you may be willing to accept lower
purity to sort the maximum possible number of target cells.
226
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 7-4 Sort Setup Window
The following is a brief description of the options in the Sort Setup window:
•
Sort Gate—Displays the list of logical gates and allows you to select a sort gate.
Selecting No Gate (all cells) allows you to acquire without sorting.
•
Sort Count—The number of cells to be sorted. Zero (0) is for continuous sorting.
•
Sort Mode—Select among Single Cell, Recovery, or Exclusion.
•
Aborted Cells—Select between List or No List to acquire to the computer the data
from aborted cells. Cells are classified as aborts when they cannot be reliably
measured. The most common cause of cells classified as aborts is that two cells
occur too close in time, ie, coincidence.
Sort Counters
Sort Counters are selected from the Cytometer menu to display the Sort Counters
window. Three counters are available to display a rate or an accumulation of these four
values: threshold, auxiliary, sort, and abort. Select Threshold or Auxiliary in Counter 1;
Sort or Threshold in Counter 2; and Abort or Sort in Counter 3.
The following is a brief description of the four values available for selection:
Sorting
•
Threshold Rate or Total—Displays the rate (events/sec) or the total number of
events triggering the threshold detector including aborted events.
•
Auxiliary Rate or Total—Displays the rate or the total number of cells that the
flow cytometer is processing including aborted cells.
337452 Rev. A
227
•
Sort Rate or Total—Displays the rate (events/sec) or the total number of sorted
cells.
•
Abort Rate or Total—Displays the rate (events/sec) or the total number of
aborted cells.
Preparing Collection Tubes
Collection tubes must be coated with a bovine serum albumin (BSA) solution to help
maintain cell integrity and increase cell yield during centrifugation. Recovery and
viability of sorted cells will be reduced if you do not coat the collection tubes. The BSA
should be filtered through a 0.45-µm filter before use. Coat the collection tubes with
4% BSA in 1X PBS.
1 Fill one to three 50-mL conical tubes with a 4% BSA (in 1X PBS + 0.1% NaN3).
Store remaining BSA at 2° to 8°C.
2 Place the tubes on ice or in the refrigerator for at least 1 hour.
3 Pour the BSA solution from the tubes into a bulk container or into other conical
tubes.
4 The BSA solution may be recycled for 1 month.
Sorting Aseptically
If you wish to perform a sterile sort, refer to the FACSort User’s Guide or FACSCalibur
System User’s Guide for detailed information on sterilizing the cytometer for an aseptic
sort.
Sorting Viable Cells
Sterile, filtered (0.22 µm) phosphate-buffered saline (PBS, pH 7.2–7.4) should be used
as sheath fluid when sorting viable cells.We do not recommend commercial sheath fluid
for sorting cells because it can affect cell viability.
228
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Sorting Practice
Sorting cells on a flow cytometer is a multistep process. The following figure outlines
the main steps involved. In this module, we will focus on starting up the system,
optimizing instrument settings, sorting, and acquiring and analyzing pre- and post-sort
data.
Set Up Sort/
Sort
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
The following exercise is an example of how a sort can be performed. It illustrates
continuous sorting without acquiring to a data file. Pre- and post-sort data is collected
to determine sort purity.
In this exercise you will:
•
Start up the system, including installing a sheath tank with appropriate fluids for
sorting and priming the sort lines
•
Optimize the cytometer for the cells of interest
•
Create a sort gate
•
Collect a pre-sort data file
•
Make selections in the Sort Setup and Sort Counters windows
•
Sort cells
•
Concentrate the sorted cells
•
Clean the sort lines
•
Collect a post-sort data file
•
Verify sort purity
Starting Up the System
Perform system startup, as described in the BD FACSCalibur System module.
NOTICE Install a sheath tank containing appropriate fluids for your sort. For example,
sterile PBS. Do not use BD FACSFlow sheath fluid if you want to sort viable cells.
Sorting
337452 Rev. A
229
Priming the Sort Lines
You will purge the sort lines to check that they are unblocked before sorting. If you are
sorting fixed cells, you can use DI water to check the lines. If you are sorting viable
cells, use sterile PBS.
1 Install a tube of DI water or sterile PBS on the SIP. If needed, also install a tank
containing sterile PBS in place of the sheath tank.
During the training class the sort samples provided are fixed cells. Hence, it is not
necessary to replace the sheath tank with a tank of PBS since cells are not viable.
2 Set the fluid control to RUN.
3 Install a 50-mL tube in the first collection port.
4 Press the sort-line purge button located inside the collection station.
5 Remove the 50-mL tube from the first collection port and place it in the second
collection port.
6 Press the sort-line purge button.
7 Remove the 50-mL tube from the second collection port and place it in the last
collection port.
8 Press the sort-line purge button.
9 Set the fluidics control to STNDBY.
Performing Quality Control
Perform quality control, as described in the BD FACSComp module.
Optimizing Settings
Before beginning your sort, you will optimize instrument settings for your cells of
interest, as well as create a sort gate.
It is assumed that you are already familiar with the optimization process. If necessary,
review the BD CellQuest Pro Software module for details about optimization before
performing the following steps.
1 Launch BD CellQuest Pro software.
2 Choose Acquire > Connect to Cytometer.
230
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
3 Open the Acquisition document you saved in the BD CellQuest Pro Software
module.
4 Load the instrument settings you saved after optimization in the BD CellQuest
Pro Software module.
Alternatively, you can get instrument settings from a previously collected data file
or use the Calib file settings.
5 Install the isotype control tube on the SIP and verify that instrument settings are
optimized.
During the training class a tube containing IgG1 FITC and IgG2a PE is provided to
check the following settings are optimized for the lysed whole blood sample you
will sort.
•
FSC and SSC
•
FSC threshold
•
Gate surrounds population of interest
•
FL1 and FL2 PMT voltages
6 Install the compensation control tube on the SIP and verify that compensation
settings are optimized.
During the training class a tube containing CD3 FITC and CD19 PE is provided
to check FL2 –%FL1 and FL1 –%FL2 compensation settings are optimized for
the lysed whole blood sample you will sort.
Creating a Sort Gate
1 Install the sort tube on the SIP.
2 Click the Polygon-Region tool in the Tool Palette.
3 Draw a region around the FITC and PE double-positive cell population in the FL1
vs FL2 plot.
This is a second region, R2. The first region (R1) around the lymphocytes was
created during optimization performed in the BD CellQuest Pro Software
module.
Sorting
337452 Rev. A
231
4 Click Pause and Abort.
5 Choose Gates > Gate List.
The Gate List window appears. You will now change the definition of G3.
6 Double-click on R3, enter R1 AND R2, and press the Return key.
Once the gate is defined in the window, the label and definition will switch from
italics to plain text. An event that satisfies both R1 and R2 will satisfy the gate
G3. A cell which is a lymphocyte and also FL1 and FL2 double positive will be
sorted.
7 Click the close button in the Gate List window.
232
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Collecting a Pre-Sort Data File
You will collect a data file of your sample before sorting. You will compare this data file
later to a data file of your sorted sample to determine the purity of the sorted sample.
1 Choose a folder and enter a file name for your sample in the Browser.
2 Deselect the Setup checkbox in the Acquisition Control window or in the
Browser.
3 Acquire a data file of the sort sample.
4 Place a tube of distilled water on the SIP and let it run for at least 30 seconds.
5 Set the fluid control to STNDBY.
6 Save the Experiment Document.
Making Selections in Sort Setup and Sort Counters
In this part of the exercise, you will select the options and conditions for your sort.
1 Choose Acquire > Sort Setup.
The Sort Setup dialog appears.
2 Make the following selections in the Sort Setup dialog.
3 Click OK.
4 Choose Cytometer > Sort Counters.
The Sort Counters window appears.
Sorting
337452 Rev. A
233
5 Choose Threshold Total, Sort Total, and Abort Rate in the Sort Counters dialog.
Sorting the Population of Interest
1 Make sure the Setup box is checked in the Acquisition Control window or in the
Browser.
When the Setup box is deselected, cells are sorted without saving data to a file.
2 Install BSA-coated 50-mL conical tubes from left to right in the collection station.
CAUTION: It takes approximately 9 minutes to fill one 50-mL tube. At any time
during the sort, clicking Pause, then Restart, will reset the counters to 0. Clicking
Pause, then Resume, will not reset the counters. This is important so that the
collection tube does not overflow. Also, when you click either Resume or Restart,
sorting will begin again starting in the leftmost collection port. If necessary,
remember to place a new BSA-coated tube in the leftmost collection port before
hitting Resume or Restart.
3 Make sure LO flow rate is selected on the instrument.
4 Set the fluid control to RUN.
5 Install the sample tube onto the SIP.
6 Click Acquire in the Acquisition Control window or in the Browser.
7 When sorting is complete, click Pause then Abort.
8 Remove the sample from the SIP. Place a tube of 1 mL of distilled water on the
SIP.
9 Set the fluid control to STNDBY.
10 Remove collection tubes from the collection station.
234
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Concentrating the Sample
NOTICE Recovered cells will have a large amount of BSA present. If your application
(eg, western-blot analysis) requires low levels of extraneous protein, resuspend the cells
in a small volume (about 1 mL) of PBS after centrifuging, transfer to a clean tube, add
PBS to wash, and centrifuge again.
1 Remove the collection tube(s) from the instrument and cap them.
2 Spin the tubes at 300 x g for 5 minutes.
3 Aspirate the supernatant.
Do not disturb the pellet.
4 Vortex the pellet and resuspend it with 0.5 mL of PBS.
5 Transfer suspension to a 12 x 75-mm BD Falcon™ test tube.
Cleaning the Sort Lines
After sorting is completed, clean the sort lines with the 30-mL syringe provided.
WARNING: If working with biohazardous material, perform the cleaning procedure
described below first with 1:10 bleach solution, followed by twice the volume of distilled
water.
Preparing the Syringe
1 Disconnect the tubing from the syringe by twisting the luer end counterclockwise.
2 Fill the syringe with distilled water.
Place the syringe nozzle in a container filled with distilled water and slowly pull
out the plunger until the syringe is full.
3 Bleed any air out of the syringe by holding it luer-end up and gently pushing in
the plunger.
4 Reconnect the tubing to the syringe by turning clockwise.
5 If necessary, bleed any air out of the tubing by pressing the tip of the quickdisconnect against the side of a beaker while gently pushing on the syringe
plunger.
6 Disconnect the upper tubing of the sheath filter from the SALINE FILTER port by
squeezing the metal clip on the quick-disconnect and pulling the connector from
the fitting.
Sorting
337452 Rev. A
235
7 Connect the tubing from the syringe to the port labeled SALINE FILTER.
Push firmly until you hear a click.
VENT VALVE
PRESS TO RELIEVE
PRESSURE
J69
J70
WASTE
SHEATH
SALINE
FILTER
Performing the Clean
1 Install a 50-mL tube in the leftmost position in the collection station.
2 Place a tube of distilled water on the SIP.
3 Set the fluid control to RUN.
4 Press the sort-line purge button.
5 Slowly yet firmly, apply pressure to the syringe plunger.
Depress the plunger until approximately 10 mL of fluid has been dispensed from
the syringe and has entered the collection tube.
NOTICE Once the button is pressed, the valve remains open for approximately 30
seconds. Press the button again if you have not dispensed all 10 mL of fluid.
6 Remove the collection tube from the leftmost collection port and place it in the
middle collection port.
7 Repeat steps 4 and 5.
8 Remove the collection tube from the middle collection port and place it in the
rightmost collection port.
9 Repeat steps 4 and 5.
236
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
10 Remove the syringe and reconnect the sheath filter tubing.
11 Choose Acquire > Sort Setup.
12 Select No Gate from the Sort Gate pop-up menu in Sort Setup.
13 Click OK.
14 Leave the instrument in STNDBY mode and install a tube of 1 mL of distilled
water on the SIP.
Collecting a Post-Sort Data File
NOTICE Before collecting the post-sort data file, run distilled water for 3 minutes. This
will ensure that carryover cells from the pre-sort sample tube will not contaminate the
post-sort analysis.
1 Collect a data file of your sorted sample.
2 Reformat the FSC vs SSC and FL1 vs FL2 acquisition plots to analysis plots.
3 Make sure both plots display ungated data.
If needed, select the plot(s) and then choose No Gate from the Gate pop-up menu
in the Inspector.
Verifying Sort Purity
You will check the sort purity of the sorted sample. The data should show that the
sorted population is at least 95% of the total events.
1 Display the post-sort data file in both plots.
2 Choose Gate Stats for either plot.
3 Evaluate the stats for %Gated or %Total for G3 (they will be the same).
Percent gated or total events should be greater than 95.0%. This is the purity of
the sort and will depend largely on sample handling.
NOTICE BSA particles might appear in your data, especially if you did not wash
the sample after concentrating it. The BSA particles generally appear on the
diagonal when seen in plots displaying fluorescence parameters, such as FL1 vs
FL2. Exclude the BSA events from your purity calculation.
Sorting
337452 Rev. A
237
Sorting Hints
Users have found that several factors should be considered to obtain the best sorting
performance from their instruments. This section compiles many of these factors for
easy reference.
Sheath Fluid
•
Always tighten the sheath tank cap before sorting.
During sorting, the sheath flow rate is important for sort timing. A poorly
tightened sheath tank cap or large amounts of air in the sheath filter could alter
the flow rate, resulting in poor sorting performance.
•
After tightening the sheath tank cap, listen for a hissing noise. Loosen and
retighten the cap if this occurs. If the hissing continues, it indicates there is a small
hole in either the cap or the tank and one or both need to be replaced.
•
Begin sorts with a full sheath tank to avoid refilling during a sort.
•
The tanks can be monitored using the Status window in BD CellQuest Pro
software, but it is better to check the level before you start an extended run.
Cell Recovery and Viability
238
•
Store tubes of sorted cells on ice if they cannot be spun immediately after
collection.
•
If viability is a concern, centrifuge cells immediately after collection and place
concentrated cells on ice in a protein-supplemented medium.
•
Viability might also improve if 5 mL of chilled media with 20% serum is placed in
each BSA-coated collection tube prior to sorting.
•
Ensure that all the sorted cells are collected from the sort lines, especially if you
are sorting for a short time.
•
At the end of the sort, remove the sample tube, install a tube of PBS on the SIP,
and restart or continue sorting for 20 seconds so that all sorted cells reach the
collection tube. The timer in BD CellQuest Pro software can be used to measure
the 20 seconds if you do not select Pause.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Sorting
•
Optimize the threshold level for your sample.
Events below the threshold level will not trigger the instrument. Events that do
not trigger the system cannot be excluded by the sort logic. For example, if red
cells and platelets are in your sample and must be rejected from the sorted
fraction, the scatter threshold level must be low enough to allow them to be
detected.
If the threshold level is too low, the instrument will detect debris, and the
coincidence of debris with your target cell could prevent the cell from being
sorted.
•
Use the LO flow rate for sorting.
The sorting specifications have been validated at this flow rate. Purity using the
HI and MED flow rates may not be equivalent.
•
Check your sort lines and the collection station electronics if the sorted sample
does not reach the collection station when sorting.
Small salt crystals may form in the sort tubing or in the collection station tubing.
They can usually be loosened easily by massaging the tubing gently. If not, use the
clog removal method described in the instrument user’s guide, or the alternate
method in the cleaning section of this training manual.
NOTICE When the lid of the BD FACSort instrument is lifted to check the sort
lines, the laser will turn off automatically as a safety measure. Check your status
window to be sure the laser has returned to full power before attempting to sort.
If the sorted sample still does not reach the collection station, check the electronic
board behind the collection station. There should be two red lights lit when the
instrument is sorting. One indicates that the instrument is in sort mode, the other
shows which collection station is active. When collection stations are switched by
removing the tube from the active station, one light should change and the other
should remain lit. If these lights are not on or do not switch properly, there is an
electronic problem with your collection station. Contact the Customer Support
Center.
•
Vortex your sample occasionally during long sorts to avoid sample settling.
Sample settling is most often noticed as a decrease in the event rate during the
sort. If the sample is removed from the sample injection port for vortexing, we
recommend that acquisition and sorting be stopped by hitting the Pause button
on the Acquisition Control window or in the Browser. Wait a few seconds after
replacing the tube before selecting the Restart or Resume buttons to avoid sorting
Sorting
337452 Rev. A
239
cells while the sample tube is pressurizing. If you wish to continue a sort that has
been paused without restarting the counters, select Resume rather than Restart.
NOTICE Before selecting Resume or Restart, you must replace any filled or
partially filled tubes in the collection station with empty BSA-coated tubes to
avoid overfilling.
•
Reset the Collection Station to tube 1 after tube 3 is full.
After you have filled all three tubes, install three empty BSA-coated tubes in the
collection station. Select Pause, then Restart or Resume. Again, the counters are
not reset if you select Resume, but are reset to 0 if you select Restart.
Clogs
Use this procedure to remove a major clog in the sort or collection station lines.
First test
•
Make sure a tube is on the SIP and the fluid control is set to RUN.
•
Open the top of the instrument and disconnect the sort line that leads down into
the valve block on the collection station.
NOTICE Do not disconnect the tubing above the flow cell off the catcher tube, as it
is very hard to reconnect and might result in a service call.
If sheath fluid comes out of this line when it is disconnected, there is no clog in
this line. You should reconnect the line and proceed to the second test described
below.
If sheath fluid does not come out, this line is clogged. Examine the line for visible
particles (probably salt crystals). Gentle pressure on the outside of the line at the
point of the clog sometimes dislodges it.
Second test
Check the individual collection station port lines leading from the valve block to the
collection tubes.
240
•
Disconnect the line to port 1 at the valve end.
•
Attach an 18-gauge luer stub to a 10-mL syringe filled with DI water.
•
Fit the needle stub into the end of the tubing.
•
Apply pressure on the syringe barrel, forcing water into the tubing and out port 1.
Repeat on the remaining two port lines.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
If this works, reconnect all the tubing and try purging the lines as described in
Priming the Sort Lines on page 230. If you cannot find a clog or dislodge one that
you do find, contact the BD Biosciences Customer Support Center.
Testing Sorting Performance
To ensure optimal performance of the instrument, periodically check the sort purity
performance.
NOTICE We recommend that BD CaliBRITE beads be diluted in BD FACSFlow
solution and run with BD FACSFlow solution as sheath fluid when performing test
sorts with BD CaliBRITE beads. If PerCP-Cy5.5 BD CaliBRITE beads are used, dilute
and run the beads using the Bead Dilution Buffer.
1 Prime the sort lines.
2 Create a sort gate.
•
Install a mixture of unlabeled, FITC, and PE BD CaliBRITE beads.
•
Create region R1 around the singlet bead population in FSC vs SSC and
region R2 around the FITC-positive population on an FL1 vs FL2 dot plot.
•
Define G3 = R1 and R2 in the Gate List and select this as the sort gate.
3 Sort into a 50-mL conical tube.
The tube is not required to be coated for this test.
4 Centrifuge the tube and resuspend the sorted cells.
Aspirate all except 0.5 mL of the supernatant, and resuspend the pellet.
5 Acquire a post-sort data file of this sample.
Sorting
•
Using the same document, reformat the FSC vs SSC and FL1 vs FL2 plots to
analysis plots.
•
Verify both plots display ungated data.
•
Display the post-sort data file in both plots.
•
Select Gate Stats for either plot.
•
Evaluate the statistics for either %Gated or %Total for G3 (they will be the
same).
337452 Rev. A
241
•
Analyze the file of sorted beads.
Percent gated or total events should be greater than 95%. This is the purity of
the sorted sample and will depend largely on sample handling technique.
Ending a Sort
The following table describes the different options you can select to end a sort.
Selection
Acquisition
Control
window
Sort Setup
window
Acquisition &
Storage
window
Click Pause,
then Abort
Outcome
Sorting ends.
Setup box
checked
Sort Count set
to zero (0)
Sort continuously. (To end the
sort, the operator must manually
click Pause, then Abort). No data
file is saved.
Setup box
checked
Sort Count
specified
Sort ends when Sort Count
specified is reached. No data file is
saved.
Setup box
unchecked
Sort Count set
to zero (0)
Event Count
specified in
Collection
Sort ends when specified Event
Count is reached. A data file will
be saved.
Criteria field
242
Setup box
unchecked
Sort Count set
to zero (0)
Event Count
or Time
specified in
Collection
Criteria field
Sort ends when specified Event
Count or Time is reached,
whichever comes first. If you wish
sorting to stop based on Time,
specify a large number in the
count field so time will expire
before the Event Count is
reached. A data file is saved.
Setup box
unchecked
Sort Count
specified
Event Count
specified in
Collection
Criteria field
Sort ends when either criteria is
reached first. A data file is saved.a
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Selection
Acquisition
Control
window
Sort Setup
window
Setup box
unchecked
Sort Count
specified
Acquisition &
Storage
window
Event Count
or Time
specified in
Collection
Criteria field
Outcome
Sort ends when specified Sort
Count, Event Count, or Time is
reached, whichever comes first. If
you wish sorting to stop based on
Time, set the Sort Count to zero
and specify a large number in the
Event Count field so time will
expire before the Event Count is
reached. A data file is saved.a
a. If the Sort Count criteria is reached before Event Count or before Time, data acquisition will also end
Sorting
337452 Rev. A
243
244
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
8
BD FACS Loader
After completing this module you will be able to:
•
Operate the BD FACS Loader using BD WorklistManager and
BD LoaderManager software.
•
Identify the major components of the Loader hardware.
•
Perform Loader troubleshooting and diagnostics.
•
Perform proper cleaning procedures for the Loader.
BD FACS Loader Option
337452 Rev. A
245
The FACS Loader provides automated introduction of prepared samples to
BD FACSCalibur, BD FACScan, and BD FACSort flow cytometers. As shown in the
following diagram, BD WorklistManager and BD LoaderManager software control the
Loader hardware and the software used to acquire and analyze data, such as
BD CellQuest Pro and BD MultiSET software.
Acquisition software
(BD CellQuest Pro or
BD MultiSET software)
BD WorklistManager software
BD LoaderManager software
Flow
Cytometer
BD FACS Loader
Figure 8-1 through Figure 8-4 identify the different Loader components. Refer to the
BD FACS Loader User’s Guide for details about each component.
BECTON
DICKINSON
FACSort
Loader drawer
BECTON
DICKINSO
N
ON
OFF
RACK
UP
DOWN
LIFTER
HIGH
LOW
MIXING
keypad
Figure 8-1 BD FACS Loader shown with a BD FACSCalibur cytometer
246
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
tube lifter
rack spindle
optical
sensors
drawer
Figure 8-2 Loader drawer
Note that the Loader is compatible with both the traditional Loader racks (gray) and
the newer, greenish racks labeled “Sample-Prep Ready.”
spindle hole
alignment hole
rack handle
Figure 8-3 Loader rack
BD FACS Loader Option
337452 Rev. A
247
LED display
RACK advance key—
press to advance the rack
to the right (if at position
01, will move to position
40)
SCSI cable
BECTON
DICKINSON
ON
LIFTER key—press to
raise or lower a tube
OFF
RACK advance key—
press to advance the
rack to the left (if at
position 01, will move
to position 02)
RACK
UP
DOWN
HIGH
LOW
LIFTER
MIXING
MIXING key—press and release
for low-energy mix; press for 3
sec. and release for high-energy
mix
Figure 8-4 Loader keypad control
Starting Up the Loader and Using It in Manual Mode
In this exercise you will start up the Loader and operate it using the remote keypad
control.
Starting Up the Loader
1 Turn on the cytometer before turning on the computer to ensure proper
communication between the cytometer and computer.
2 Make sure the Loader rack and cover are not on the Loader.
3 Turn on the Loader power from the Loader keypad control.
4 Check the cytometer fluid levels.
5 Fill the sheath reservoir and empty the waste reservoir, if necessary.
6 Remove the distilled water tube from the Sample Injection Port (SIP) by grasping
the bottom of the test tube and swinging the Droplet Containment Module
(DCM) arm to the left.
NOTICE If you do not grasp the tube, it will fall into the loader bucket area.
248
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Installing the Rack and Loader Cover
1 Place a rack on the Loader with distilled water tubes in positions 1 and 2.
To install a rack, position the hole in the rack handle over the center spindle of
the Loader drawer and rotate the rack until the alignment guide pin fits into the
small alignment hole at the top of the rack. See Figure 8-3 on page 247 and
Figure 8-2 on page 247.
2 Push the Loader drawer completely in.
3 Place the Loader cover on the Loader drawer.
The Loader cycles and displays a Tube 01 message on the Loader keypad control.
4 Place the cytometer fluidics in RUN mode.
Using the Loader Keypad Control
1 Using the keypad, press the MIXING key for approximately 3 seconds to activate
the high-energy mix.
2 Use the LIFTER key to install the distilled water tube in position 1 on the SIP.
If proper pressurization occurs, the RUN button indicator light turns green, and
the lifter remains in the up position. If the tube does not pressurize, a message
flashes on the keypad and an alarm beeps.
NOTICE To stop the alarm, remove and replace the Loader cover when the lifter is
completely down. If pressurization problems persist, refer to the troubleshooting
section in the BD FACS Loader User’s Guide.
1 After the RUN button turns green, lower the distilled water tube using the
LIFTER key on the Loader keypad control.
2 Use the rack advance key (
) to index the rack to position 2.
The tube position is displayed on the keypad.
3 Use the LIFTER key to install the distilled water tube on the SIP.
4 After the RUN button turns green, press the LIFTER key to lower the tube.
5 Use the RACK back key (
BD FACS Loader Option
) to index the rack to position 1.
337452 Rev. A
249
Removing the Loader Cover and Rack
1 Remove the Loader cover.
2 Remove the Loader rack.
To remove a rack from the Loader, slide the Loader cover forward and pull the
Loader drawer out. Place your thumb on the center spindle that protrudes
through the top of the rack handle. Press down with your thumb while gently
pulling up on the handle with two fingers, as illustrated in the following figure.
3 Install a distilled water tube on the SIP, with the support arm underneath.
4 Place the cytometer fluidics in STANDBY mode.
Preparing Documents
You need to prepare a BD CellQuest Pro Experiment document or BD MultiSET
Schedule document or both before you use the BD FACS Loader for the first time. You
can also prepare a BD Worklist Manager software worklist template. You need to
prepare these documents only once and then reuse them, as needed.
Preparing a BD CellQuest Pro Experiment Document for
Use with BD FACS Loader
Keep the following in mind when creating a BD CellQuest Pro Experiment document
for use with the BD FACS Loader.
250
•
Create Acquisition to Analysis type plots if you want to print a copy of the data
just acquired after each tube is acquired.
•
Specify a time limit in Acquire > Acquisition and Storage.
•
Create a panel and then select the panel in the Acquisition Browser.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
•
Save the Experiment document in the CellQuest Experiments Folder
(BD Applications/WorklistManager Folder/CellQuest Experiments Folder).
•
Save instrument settings you might want to use in the Instrument Settings folder
(BD Files/Instrument Settings Files).
•
Specify tube-specific settings, if needed. See Specifying Tube-Specific Settings
(Optional) on page 99 in the BD CellQuest Pro Software module for details.
In the following exercise, you will modify an Experiment document you created
previously. If you are not familiar with BD CellQuest Pro Software, complete the
BD CellQuest Pro Software module before performing the following exercise.
1 Open the BD CellQuest Pro Experiment document you saved earlier in the week.
Use the Experiment document that you created to acquire immunophenotyping
samples.
2 Format all dot plots to Acquisition -> Analysis plots.
•
Select all the plots in the document.
•
Choose Windows > Show Inspector.
•
Choose Acquisition -> Analysis from the Plot Type pop-up menu in the
Inspector.
3 Double-click the Global Acquisition & Storage Settings icon in the Acquisition
Browser.
The Inspector appears.
4 Make changes in the Acquisition & Storage Inspector.
For this exercise, make the following changes. See Figure 8-5 on page 252.
•
Change the Collection Criteria from Event Count to Event Count or Time.
•
In the Acquisition will stop when field, enter 2500 and choose G1 from the
pop-up menu.
This will ensure that at least 2500 events in G1 are counted before
acquisition ends.
•
In the seconds field, enter 420.
Acquisition will end after 420 seconds if the event count has not been satisfied.
NOTICE For walkaway operation, enter a time less than 600 seconds (10 minutes)
for the stopping time. If you choose Event Count only or enter a time of more
BD FACS Loader Option
337452 Rev. A
251
than 10 minutes, you run the risk of BD WorklistManager software timing out if
the specified number of events is not collected during the entered time, or after
10 minutes. During a timeout, no data is saved, and you might not have a
sufficient amount of sample left to rerun.
Figure 8-5 Acquisition & Storage Inspector
5 Choose the panel you created previously.
Load a panel in the Acquisition Browser.
6 Choose File > Save Document As.
7 Enter a name for the document.
For example, enter Loader Exercise.
8 Navigate to the BD Applications/WorklistManager Folder/CellQuest Experiment
Folder.
9 Click Save.
All BD CellQuest Pro Experiment documents must be saved in the CellQuest
Experiments Folder if you want to use them for data acquisition and analysis with
the Loader.
252
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
10 Quit BD CellQuest Pro software.
11 Proceed to one of the following.
•
Modifying the Acquisition Tube List (Optional).
•
Defining Tube-Specific Settings (Optional) on page 254.
•
Using BD CellQuest Pro Software with the Loader on page 257.
Modifying the Acquisition Tube List (Optional)
You can modify the order of the tubes to be acquired in the Acquisition view of the
Browser, if necessary.
1 Make sure the Acquisition view of the Browser is displayed.
2 Make necessary changes to the tube list, if necessary.
To move a tube, click on the tube icon and drag to the position you want on the
list.
NOTICE Do not delete tubes or add tubes to the list in the Acquisition Browser. To
add or remove tubes from the list, choose Edit Panels from the Acquire menu and
modify the panel you want to change. Adding or deleting tubes from the
Acquisition Tube List in the Browser can affect acquisition in
BD WorklistManager software.
3 Make changes to tubes, if needed.
For example, you can change the tube name and parameter labels using the
Inspector. To make changes to a tube do the following.
•
Click the tube icon in the Browser and press a-I.
•
Make changes in the Inspector.
Changes appear after you exit the field (eg, after pressing Tab).
4 Save the Experiment document.
5 Proceed to Defining Tube-Specific Settings (Optional) on page 254 or to Using
BD CellQuest Pro Software with the Loader on page 257.
BD FACS Loader Option
337452 Rev. A
253
Defining Tube-Specific Settings (Optional)
You can define one or more of the following tube-specific settings, if needed.
•
acquisition and storage settings
•
instrument settings
•
associating plots with tubes (only available with panels)
See Specifying Tube-Specific Settings (Optional) on page 99 in Module 4 BD CellQuest
Pro Software for details.
For this exercise, try the following.
1 Define tube-specific acquisition and storage settings for the second tube in the
tube list in the Browser.
Instead of 2500 of G1 events, change it to 2500 of All events.
2 Define tube-specific instrument settings for the third tube in the tube list in the
Browser.
Choose the Calib file in the BD Files folder.
3 Save the Experiment document.
4 Continue with preparation of a BD MultiSET Schedule Document or proceed to
Using BD CellQuest Pro Software with the Loader on page 257.
Preparing a BD MultiSET Schedule Document for Use
with BD FACS Loader
Keep the following in mind when creating a BD MultiSET Schedule document for use
with the BD FACS Loader.
254
•
A predefined Schedule document (MultiSET.WM.sch) is available in the
MultiSET folder. You can use the document as defined, or make modifications
before using it, if needed.
•
Specify Physician, Laboratory, and Summary Report choices in the Test Prefs
view.
•
In the Lot IDs dialog (Tools > Lot IDs), specify lot IDs and beads/pellet for
BD TruCOUNT beads.
•
In Reagent Tools dialog (Tools > Reagent Tools), create user-defined reagents, if
needed.
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
•
In the Subset Ranges dialog (Tools > Subset Ranges), create user-defined ranges, if
needed.
•
In the Panel Tools dialog (Tools > Panel Tools), create panels you might need.
•
In the MultiSET Preferences dialog (MultiSET > Preferences), specify preferences
for export, printing, panels, events, LJ MSET Data, columns, banners, and
comments.
•
Save instrument settings you might want to use in the Instrument Settings folder
(BD Files/Instrument Settings Files).
In the following exercise, you will modify the MultiSET.WM.sch Schedule document. If
you are not familiar with BD MultiSET Software, complete the BD MultiSET Software
module before performing the following exercise.
1 Navigate to the MultiSET.WM.sch file in the BD Applications/MultiSET Folder
and double-click to open it.
2 Enter necessary information in the Sign In view.
The operator ID is required.
3 Click Accept.
4 Verify the From Cytometer: Acquisition with Analysis radio button is selected in
the Set Up view.
5 Click Accept.
6 Click Skip FACSComp.
7 Make the following selections in the Test Prefs view.
•
Verify all the subset boxes are selected under Physician Report in the Test
Prefs view.
•
Select Report Reference Ranges.
BD FACS Loader Option
337452 Rev. A
255
•
Select both Report Percents and Report Absolute Counts under Laboratory
Report Choices.
•
Select Sample Name under Summary Report ID.
8 Click Subset Ranges, Panel Tools, or Reagent Tools to make any changes in those
windows, if needed.
9 Click Lot IDs.
10 Enter Beads/Pellet value and Lot ID for BD TruCOUNT tubes, if needed.
Always be sure to enter the correct BD TruCOUNT tube lot ID and bead count
when obtaining absolute counts.
11 Click Save.
12 Make selections in MultiSET > Preferences, if needed.
13 Choose File > Save As.
14 Enter a name for the Schedule document, choose a folder destination, and then
click Save.
You do not have to save the Schedule document to a specific location. During the
training week, save the document to your folder in the Student folder.
15 Quit BD MultiSET Software.
16 Proceed to Using BD MultiSET Software with the Loader on page 270.
256
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Using BD CellQuest Pro Software with the Loader
Acquisition of data using the Loader is a multistep process. The following figure
outlines the main steps involved. In this module, we will focus on optimization of
instrument settings, data acquisition, and shutdown.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
In this exercise, you will do the following.
•
Optimize instrument settings
•
Define conditions for data acquisition and storage
•
Acquire data
Before beginning this exercise, perform the following procedures.
•
System Startup on page 20 in the BD FACSCalibur System module.
•
Performing Instrument QC and Setup with the BD FACS Loader Option on
page 62, as described in the BD FACSComp Software module.
Optimizing Instrument Settings
In this exercise, you will optimize instrument settings as outlined in the following
graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Set up an Experiment document.
• Optimize instrument settings for your sample type.
• Save optimized settings.
To perform optimization, you can use the Optimization view in BD FACSComp
software or optimize in BD CellQuest Pro software.
NOTICE
Do one of the following optimization exercises.
Optimizing in BD FACSComp Software
1 Launch BD FACSComp software if it is not already opened.
2 Click the Optimization icon.
BD FACS Loader Option
337452 Rev. A
257
3 Select Optimization 5: CellQuest.opt from the pop-up menu.
4 Place your optimization tubes in the Loader rack in the positions indicated in the
following table.
Tube
Position
Isotype control
1
FITC control
2
PE control
3
PerCP control
4
APC control
5
5 Choose Load Tube #1 from the tube pop-up menu.
This will lift the isotype control tube onto the SIP.
6 Click Start.
7 Perform the first four optimization steps.
•
Adjust FSC and SSC PMT to move the population in the Gate Plot.
•
Adjust FSC Threshold to remove most of the debris.
•
Place a gate on the population of interest by clicking Live Gate and drawing a
polygon on the Gate Plot.
•
Adjust the FL1, FL2, and FL3 PMTs to place the populations in the bottom
left corner of the plots.
NOTICE The two plots to the right of the Gate Plot automatically display only
events that are found in the gate created.
8 Choose Load Tube #2 from the tube pop-up menu.
This lowers the first tube and installs the next tube onto the SIP.
9 Check compensation by adjusting the appropriate compensation networks.
258
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
10 Continue with subsequent tubes until all compensation networks have been
checked.
Click on the parameter label to change the parameter on the pop-up menu, if
necessary.
11 Click Save Optimized Settings after compensation is complete.
This saves the instrument settings with the designated file name to
BD Files/Instrument Settings Files Folder.
NOTICE You can customize your optimization setups by going to the FACSComp
menu and selecting Preferences. Select Optimization in the Preferences List and
make your choices. Refer to the BD FACSComp Software User’s Guide for more
details.
12 Click Quit to exit BD FACSComp software.
13 Continue with Acquiring Data on page 260.
Optimizing in BD CellQuest Pro Software
1 Open the Experiment document you created previously in the BD CellQuest Pro
Software module.
2 Optimize instrument settings.
Use the isotype, FITC, PE, PerCP, and APC controls to perform the
5 optimization steps. See Optimizing Instrument Settings on page 78 in the
BD CellQuest Pro Software module for details.
3 Save the optimized instrument settings in BD Files folder/Instrument Settings Files
folder.
4 Quit BD CellQuest Pro software when you are finished.
BD FACS Loader Option
337452 Rev. A
259
Acquiring Data
In this exercise, you will acquire data as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Specify data collection information (file name, storage location, sample ID, and so on).
• Specify rack ID.
• Click Acquire.
Entering Information in BD WorklistManager Software
In this part of the exercise, you will create and save a BD WorklistManager software
worklist document and enter all necessary information in the following views.
•
Sign In
•
Set Up
•
Preferences
•
Worklist
BD Worklist Manager software will not launch if other BD applications are already
running. In addition, while BD Worklist Manager software is running, other BD
applications cannot be launched independently.
1 Verify all BD applications are closed.
Look at the BD application icons in the Dock and make sure that they are all
closed. An opened application has a black triangle beneath it.
2 Launch BD Worklist Manager software by clicking the application icon in the
Dock.
Worklist
Manager
Loader
Manager
Entering Information in the Sign In and Set Up Views
When BD Worklist Manager software is launched, the main application window opens
and the software also automatically launches BD Loader Manager software and checks
to see if the Loader is connected.
260
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Sign In View
menu bar
icon bar
message box
content area
button panel
Figure 8-6 Sign In view
The LoaderManager Status window appears, displaying connectivity status and
whether the Loader is controlled manually or by the computer.
Figure 8-7 Loader Status dialog
Note that BD Loader Manager software can also be launched independently of
BD Worklist Manager software in order to access additional features. Refer to the
BD FACS Loader User’s Guide for details.
1 Fill in the Operator field.
Only the Operator field is required. Entries in Institution and Laboratory fields
are optional.
2 Click Accept.
BD FACS Loader Option
337452 Rev. A
261
Set Up View
•
The selection under File Name Prefix determines the file name prefix for data files
saved. You must also enter this information at the Worklist view.
•
The selection under Summary Report Sample Label determines the label for
samples on the Summary Report.
•
When the Summary Report checkbox is checked under Worklist, a Summary
Report is saved.
•
Template is disabled when CellQuest is the Assay Type selected under Automatic
Savings Options. When MultiSET is the Assay Type chosen, you can specify a
BD MultiSET software Schedule document to use when acquiring data on the
Loader.
•
The selections under Automatic Savings Options determines whether data,
export, and report files are saved.
1 Make the following selections in the Set Up view.
262
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Making Selections in Assay Settings
Next, you will link instrument settings to panels and specify mix and printing settings.
This ensures that appropriate instrument and mix settings are used when you run a
particular panel. Only Experiment documents saved in BD Applications folder/
WorklistManager Folder/CellQuest Experiment Folder are listed under the Panel
column. Only instrument settings saved in BD Files folder/Instrument Settings Files
folder are listed under the Instrument Settings column.
1 Click Assay Settings in the Set Up view.
2 Click the CELLQuest icon in the Assay Settings dialog.
CELLQuest icon
instrument
settings list
3 Link an instrument settings file with the panel you created.
For example, choose the CellQuest.opt instrument settings.
panel name
Experiment document name
Each panel can be linked to only one instrument settings file at a time. Note that
the names listed under Panel are panel name: Experiment document name.
BD FACS Loader Option
337452 Rev. A
263
You can resize the Panel or Instrument Settings column by placing the cursor on
the vertical line between the two columns until you see a double-sided arrow.
Drag the arrow to resize the columns.
4 Choose Mix Settings.
•
Acquisition Start Delay: Specify 3 seconds.
•
Start-of-Rack-Mix: Specify 10 seconds.
•
Interim Mix: Specify 1 sec and Every 5 min.
5 If it is not already checked, click in the Print CellQuest Plots checkbox.
After each tube in a panel is acquired, a copy of the BD CellQuest Pro document
will print.
NOTICE In the BD CellQuest Pro document, if you created plots that are linked to
specific tubes in a panel, only the printout from the last tube in the panel will
contain plots with data displayed in all plots.
Additionally, options chosen in the Page Setup and Print dialog boxes within
BD WorklistManager software apply only to the Summary Report and Rack
Manifest printouts.
6 Click Save.
A dialog box appears.
7 Click OK.
Saving a Worklist
Before entering any sample information, you will save information entered so far. The
saved file is a BD WorklistManager worklist that you can use as a template to acquire
data on a daily basis, as needed.
TIP Once saved, make the worklist a stationery pad or a locked document. When you
open a worklist that is a stationery pad, a copy of the original opens. A locked document
prevents the original worklist from being accidentally overwritten.
264
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
1 Choose File > Save.
2 Enter a name for the file and choose a folder destination.
During the training class, choose your folder in the Student folder.
Entering Information in the Worklist View
Before you can acquire data, you need to enter sample information in the Worklist view
in BD WorklistManager software. You can enter most of the information manually, or
you can import a saved worklist from the BD FACS Sample Prep Assistant (SPA). Refer
to the BD FACS Loader User’s Guide for details.
You must enter all necessary information to fulfill the selections made in the Set Up
view. For example, if you selected Sample Name as the prefix in the File Name Prefix
option in the Set Up view, you must enter information in the Sample Name column in
the Worklist view.
1 Click the Accept button in the Set Up view.
The FACSComp view appears.
2 Click Skip FACSComp.
The Worklist view is displayed.
status
icon
Figure 8-8 Worklist view
The status icons located to the left of the entry numbers indicate whether or not
all necessary sample information has been entered.
BD FACS Loader Option
337452 Rev. A
265
The icons also provide the acquisition status of each sample.
3 Enter one sample and choose the appropriate information.
Enter the following information for the sample.
•
Sample ID: Joe Smith (note that the icon changes).
•
Assay: Choose CellQuest.
•
Panel Name: Choose the appropriate panel (your 3-color OR 4-color panel).
Note that the icon changes to a rack icon.
NOTICE If you routinely run a particular assay and a particular panel with that
assay, you can choose the fixed assay and the fixed panel by clicking the Fixed
Assay/Panel button. Choosing a fixed assay and a fixed panel will automatically
fill the Assay and Panel columns in the Worklist view with the assay and panel
you chose. For more information about the Assay and Panel selections, refer to
the FACS Loader User’s Guide.
Assigning Rack ID
Positions 39 and 40 are usually reserved for bleach and distilled water tubes and are
used for the cleaning performed after the end of a Worklist run.
If patient samples take up all 40 positions on a rack, you will be prompted after the
tube in position 40 has been acquired to install a rack containing bleach and distilled
water tubes to perform the cleaning procedure. You can load the bleach tube (in
position 39) and the distilled water tube (in position 40) on any rack that has not been
previously assigned.
NOTICE All tubes in a given sample’s panel must reside on one rack. For example if
you have ten patient samples, each patient sample consisting of six tubes (60 tubes
total), tubes for patient samples 1 through 6 would go onto one rack (36 tubes) and
patient samples 7 through 10 would go onto another rack (24 tubes).
1 Click the Assign Rack button.
The Assign Racks dialog box appears. See Figure 8-9 on page 267.
266
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 8-9 Assign Racks dialog
Note that when a sample name is highlighted in the Sample List field, you can see
the tube number, which reagent combination is assigned to each tube in the panel,
and the numerical position of the sample tubes in the rack sequence.
2 Choose the appropriate Rack ID from the pop-up menu.
3 Click the Print Rack button to print the Rack Manifest, then click OK.
The Rack Manifest contains the sample information, reagent names, and tube
locations as well as an overview of the rack with the entry numbers for each
sample. After clicking OK, the Location column in the Worklist view is filled in.
Running Samples
In this exercise, you will save the sample information you just entered. To prevent
overwriting the original worklist you saved before entering sample information, choose
Save As rather than Save.
You will also acquire your data.
1 Choose File > Save As.
2 Enter a unique name and choose a folder destination.
Again, during the training week choose your folder in the Student folder.
3 Vortex all sample tubes before placing them in the rack.
4 Load the rack with tubes, including two cleaning tubes, according to the printed
Rack Manifest.
Install a tube containing 3 mL of a bleach solution, such as BD FACS cleaning
solution, in position 39 and a tube containing 3 mL of DI water in position 40.
5 Place the rack on the Loader.
BD FACS Loader Option
337452 Rev. A
267
6 Close the Loader drawer completely.
7 Place the cover on the Loader.
The rack of tubes will spin.
8 Verify that the cytometer fluidics control is in RUN mode and choose the flow
rate.
9 Click the Run Tests button in the Worklist view.
The Loader starts its protocol.
10 When the cleaning cycle is complete, print the Summary Report by choosing File
> Print.
11 Remove the Loader cover, pull the Loader drawer out, and remove the rack.
12 Install a tube of distilled water on the SIP and place the support arm underneath
the tube.
13 Press STNDBY on the fluidics panel on the cytometer.
14 Quit WorklistManager software.
Reviewing the Summary Report
A Summary Report appears in the Summary view at the completion of every run.
Figure 8-10 on page 269 provides an example. Entries listed are not similar to those in
your Summary Report.
The Summary Report lists all sample and reagent information, the rack location, the
data files saved, and the status of the run. If an error occurred during the run, an error
message appears on the Summary Report. Refer to the BD FACS Loader User’s Guide
for assistance in troubleshooting errors.
You can save and print the Summary Report by choosing the appropriate commands
from the File menu. If you clicked the Summary Report checkbox at the Set Up view,
the report is automatically saved when the Worklist is completed or stopped.
At the Summary view you can quit the program or choose to run more samples by
clicking the Worklist icon.
268
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Figure 8-10 Summary Report
Shutting Down
If you no longer need the cytometer or computer turned on, do the following.
1 Remove the Loader rack.
2 Install a tube containing no more than 1 mL of DI water on the SIP.
3 Turn off the computer.
4 Turn off the cytometer.
BD FACS Loader Option
337452 Rev. A
269
Using BD MultiSET Software with the Loader
Acquisition of data using the Loader is a multistep process. The following figure
outlines the main steps involved. In this module, we will focus on optimization of
instrument settings, data acquisition, and data analysis.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
In this exercise, you will do the following.
•
Optimize instrument settings
•
Define conditions for data acquisition and storage
•
Acquire data
Before beginning this exercise, perform the following procedures.
•
System Startup on page 20 in the BD FACSCalibur System module.
•
Performing Instrument QC and Setup with the BD FACS Loader Option on
page 62, as described in the BD FACSComp Software module.
Optimizing Instrument Settings
In this exercise, you will optimize instrument settings as outlined in the following
graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Optimize SSC settings, if needed.
• Optimize FL3 threshold setting, if needed.
• Save optimized settings.
To perform optimization, you can use the Optimization view in BD FACSComp
software or optimize in BD MultiSET software. In this exercise, you will optimize in
BD FACSComp software.
1 Launch BD FACSComp software it is not already opened.
2 Click the Optimization icon.
270
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
The Optimization view contains five optimization setups. Choose a setup by
selecting it from the pop-up menu. Follow the appropriate instructions for the
application you are running.
NOTICE The two plots to the right of the Gate Plot display only events that are
found in the gate created.
3 Choose Optimization 1: MultiTEST.opt or Optimization 2: TriTEST.opt from the
pop-up menu.
Depending on whether you are running four-color or three-color samples, choose
MultiTEST or TriTEST, respectively.
4 Place any of the lyse/no-wash tubes on the rack in position #1.
5 Place the rack and the Loader cover on the Loader.
6 Choose Load Tube #1 from the tube pop-up menu, and click Start.
This will lift the tube up, onto the SIP.
7 Adjust the SSC PMT if necessary.
8 Adjust FL3 Threshold to exclude most of the debris.
NOTICE It is recommended that the fluorescence detectors and compensation
settings not be adjusted.
9 Click Save Optimized Settings after compensation is complete.
This saves the instrument settings with the designated file name to
BD Files/Instrument Settings Files folder.
NOTICE You can customize your optimization setups by going to the FACSComp
menu and selecting Preferences. Select Optimization in the Preferences List and
make your choices. Refer to the BD FACSComp Software User’s Guide for more
details.
10 Click Quit to exit BD FACSComp software.
BD FACS Loader Option
337452 Rev. A
271
Acquiring Data
In this exercise, you will acquire data as outlined in the following graphic.
Start Up
System
Perform
QC
Optimize
Settings
Acquire
Data
Analyze
Data
Shut Down
System
• Specify data collection information (file name, storage location, sample ID, and so on).
• Specify rack ID.
• Click Acquire.
Entering Information in BD WorklistManager Software
In this part of the exercise, you will create and save a BD WorklistManager software
worklist document and enter all necessary information in the following views.
•
Sign In
•
Set Up
•
Preferences
•
Worklist
BD Worklist Manager software will not launch if other BD applications are already
running. In addition, while BD Worklist Manager software is running, other BD
applications cannot be launched independently.
1 Verify all BD applications are closed.
Look at the BD application icons in the Dock and make sure they are all closed.
An open application has a black triangle beneath it.
2 Launch BD Worklist Manager software by clicking the application icon in the
Dock.
Worklist
Manager
Loader
Manager
Entering Information in the Sign In and Set Up Views
When BD WorklistManager software is launched, the main application window opens
(see Figure 8-6 on page 261) and the software also automatically launches
BD LoaderManager software and checks to see if the Loader is connected.
272
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
The Loader Status window appears (see Figure 8-7 on page 261), displaying
connectivity status and whether the Loader is controlled manually or by the computer.
Note that BD Loader Manager software can also be launched independently of
BD Worklist Manager software in order to access additional features. Refer to the
BD FACS Loader User’s Guide for details.
1 Fill in the Operator field in the Sign In view.
Only the Operator field is required. Entries in Institution and Laboratory are
optional.
2 Click Accept.
Set Up View
•
The selection under File Name Prefix determines the file name prefix for data files
saved. You must also enter this information at the Worklist view.
•
The selection under Summary Report Sample Label determines the label for
samples on the Summary Report.
•
When the Summary Report checkbox is checked under Worklist, a Summary
Report is saved.
•
When MultiSET is the Assay Type chosen, you can specify a BD MultiSET
software Schedule document to use when acquiring data on the Loader.
•
The selections under Automatic Savings Options determines whether data,
export, and report files are saved.
1 Make the following selections in the Set Up view.
BD FACS Loader Option
337452 Rev. A
273
Making Selections in Assay Settings
Next, you will link instrument settings to panels and specify mix and printing settings.
This ensures that appropriate instrument and mix settings are used when you run a
particular panel. Only panels previously created in BD MultiSET software are listed
under the Panel column. Only instrument settings saved in BD Files folder/Instrument
Settings Files folder are listed under the Instrument Settings column.
1 Click Assay Settings in the Set Up view.
2 Click the MultiSET icon in the Assay Settings dialog.
instrument
settings list
MultiSET icon
3 Link an instrument settings file with the panel you created.
For example, choose the TriTEST.opt instrument settings if you are running
samples prepared with BD TriTEST reagents. Choose the MultiTEST.opt
instrument settings if you are running samples prepared with BD MultiTEST
reagents.
Each panel can be linked to only one instrument settings file at a time.
274
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
You can resize the Panel or Instrument Settings column by placing the cursor on
the vertical line between the two columns until you see a double-sided arrow.
Drag the arrow to resize the columns.
4 Choose Mix Settings.
•
Acquisition Start Delay: Specify 3 seconds.
•
Start-of-Rack-Mix: Specify 10 seconds.
•
Interim Mix: Specify 1 sec and Every 5 min.
NOTICE Options chosen in the Page Setup and Print dialog boxes within
BD WorklistManager software apply only to the Summary Report and Rack
Manifest printouts.
5 Click Save.
A dialog box appears.
6 Click OK.
Saving a Worklist
Before entering any sample information, you will save information entered so far. The
saved file is a BD WorklistManager worklist that you can use as a template to acquire
data on a daily basis, as needed.
1 Choose File > Save.
2 Enter a name for the file and choose a folder destination.
During the training class, choose your folder in the Student folder.
Entering Information in the Worklist View
Before you can acquire data, you need to enter sample information in the Worklist view
in BD WorklistManager software. You can enter most of the information manually, or
you can import a saved worklist from the BD FACS Sample Prep Assistant (SPA). Refer
to the BD FACS Loader User’s Guide for details.
BD FACS Loader Option
337452 Rev. A
275
You must enter all necessary information to fulfill the selections made in the Set Up
view. For example, if you selected Sample Name as the prefix in the File Name Prefix
option in the Set Up view, you must enter information in the Sample Name column in
the Worklist view.
1 Click the Accept button in the Set Up view.
The FACSComp view appears.
2 Click Skip FACSComp.
The Worklist view is displayed. See Figure 8-8 on page 265.
The status icons located to the left of the entry numbers indicate whether or not
all necessary sample information has been entered. The icons also provide the
acquisition status of each sample.
3 Scroll through the Worklist view.
Note the Parameter #1, Parameter #2, and Parameter #3 columns. You might
need these columns to enter information such as white blood cell count (WBC),
% Lymphs, or Absolute Lymphs when determining absolute counts using
information from hematology counters. Refer to the BD MultiSET Software
User’s Guide for details.
4 Enter one sample and choose the appropriate information.
Enter the following information for the sample.
•
Sample ID: Joe Smith (Note that the icon changes).
•
Assay: Choose MultiSET.
•
Panel Name: Choose 3C TBNK + TruC or 4C TNBK + TruC.
Note that the icon changes to a rack icon.
NOTICE If you routinely run a particular assay and a particular panel with that
assay, you can choose the fixed assay and the fixed panel by clicking the Fixed
Assay/Panel button. Choosing a fixed assay and a fixed panel will automatically
fill the Assay and Panel columns in the Worklist view with the assay and panel
you chose. For more information about the Assay and Panel selections, refer to
the BD FACS Loader User’s Guide.
Assigning Rack ID
Positions 39 and 40 are usually reserved for bleach and distilled water tubes and are
used for the cleaning performed after the end of a Worklist run.
276
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
If patient samples take up all 40 positions on a rack, you will be prompted after the
tube in position 40 has been acquired to install a rack containing bleach and distilled
water tubes to perform the cleaning procedure. You can load the bleach tube (in
position 39) and the distilled water tube (in position 40) on any rack that has not been
previously assigned.
NOTICE All tubes in a given sample’s panel must reside on one rack. For example if
you have ten patient samples, each patient sample consisting of six tubes (60 tubes
total), tubes for patient samples 1 through 6 would go onto one rack (36 tubes) and
patient samples 7 through 10 would go onto another rack (24 tubes).
1 Click the Assign Rack button.
The Assign Racks dialog box appears.
Note that when a sample name is highlighted in the Sample List field, you can see
the tube number, which reagent combination is assigned to each tube in the panel,
and the numerical position of the sample tubes in the rack sequence.
2 Choose the appropriate Rack ID from the pop-up menu.
3 Click the Print Rack button to print the Rack Manifest, then click OK.
The Rack Manifest contains the sample information, reagent names, and tube
locations as well as an overview of the rack with the entry numbers for each
sample. After clicking OK, the Location column in the Worklist view is filled in.
BD FACS Loader Option
337452 Rev. A
277
Running Samples
In this exercise, you will save the sample information you just entered. To prevent
overwriting the original worklist you saved before entering sample information, choose
Save As rather than Save.
You will also acquire your data.
1 Choose File > Save As.
2 Enter a unique name and choose a folder destination.
Again, during the training week choose your folder in the Student folder.
3 Vortex all sample tubes before placing them in the rack.
4 Load the rack with tubes, including two cleaning tubes, according to the printed
Rack Manifest.
Install a tube containing 3 mL of a bleach solution, such as BD FACSClean
solution, in position 39 and a tube containing 3 mL of DI water in position 40.
5 Place the rack on the Loader.
6 Close the Loader drawer completely, and place the cover on the Loader.
The rack of tubes will spin.
7 Verify the cytometer fluidics control is in RUN mode and choose the flow rate.
8 Click the Run Tests button in the Worklist view.
The Loader starts its protocol.
9 When the cleaning cycle is complete, print the Summary Report by choosing File
> Print.
10 Remove the Loader cover, pull the Loader drawer out, and remove the rack.
11 Install a tube of distilled water on the SIP and place the support arm underneath
the tube.
12 Press STNDBY on the fluidics panel on the cytometer.
13 Quit WorklistManager software.
278
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Reviewing the Summary Report
A Summary Report appears in the Summary view at the completion of every run.
Figure 8-10 on page 269 provides an example. Entries listed are not similar to those in
your summary report.
The Summary Report lists all sample and reagent information, the rack location, the
data files saved, and the status of the run. If an error occurred during the run, an error
message appears on the Summary Report. Refer to the BD FACS Loader User’s Guide
for assistance in troubleshooting errors.
You can save and print the Summary Report by choosing the appropriate commands
from the File menu. If you clicked the Summary Report checkbox at the Set Up view,
the report is automatically saved when the Worklist is completed or stopped.
At the Summary view you can quit the program or choose to run more samples by
clicking the Worklist icon.
Shutting Down
If you no longer need the cytometer or computer turned on, do the following.
1 Remove the Loader rack.
2 Install a tube containing no more than 1 mL of DI water on the SIP.
3 Turn off the computer.
4 Turn off the cytometer.
Cleaning
This section describes how to perform a Long Clean using BD LoaderManager
software, and how to shut down the Loader. Note that a Long Clean is performed
automatically at the end of each Worklist run. Run the cleaning procedure manually if
you stop a Worklist before it is complete, or for additional, optional cleaning after the
procedure has been run automatically.
1 Quit BD WorklistManager software software, if it is open.
2 Launch BD LoaderManager software.
BD FACS Loader Option
337452 Rev. A
279
The Loader Status window appears.
3 Click the Maintenance button.
The Maintenance and Diagnostics dialog appears.
4 Verify the Long Clean option is selected; click Run.
The Long Clean procedure runs bleach and distilled water through the cytometer
for approximately 12 minutes.
A message appears, prompting you to install bleach and rinse tubes.
5 Set up a Loader rack with cleaning tubes; install the rack on the Loader.
Install a tube containing 3 mL of a bleach solution such as BD FACS cleaning
solution in position 39 and a tube containing 3 mL of DI water in position 40.
6 Click Run.
A cleaning status dialog appears.
7 When the cleaning cycle is complete, remove the Loader cover and rack.
8 Install a tube containing no more than 1 mL of DI water on the SIP.
280
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
9 Put the cytometer in Standby mode.
10 Quit BD LoaderManager software.
11 Push in the Loader drawer; leave the Loader rack and cover off.
Additional Information
It is important to review the BD FACS Loader User’s Guide for additional information
not covered in this module.
Additionally, online help is now available in BD WorklistManager software. Practice
using this feature by doing the following.
1 From the Help menu, choose BD WorklistManager Help.
The Help window appears.
The fastest way to search for help is to use the Ask a Question feature. You can
also search by looking through the table of contents or index.
2 Enter rack ID in the Search field; press Return.
A list of topics appears in the lower portion of the Help view. See Figure 8-11 on
page 282. Double-click on a topic to go to that section of Help.
BD FACS Loader Option
337452 Rev. A
281
Figure 8-11 Search Results dialog
3 When you are finished, close the Help viewer by clicking the red button.
4 Choose WorklistManager > Quit WorklistManager. Click Don’t Save.
282
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Lab Exercise: Troubleshooting
In this exercise, the instructor will perform another Loader run. This time, several
conditions have been set to mimic possible problems you might encounter during a
Loader run. You will need to observe any error messages that appear, determine what
are the problems, and solve the problems.
1 What was the first error message you encountered?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
2 Fix the problem and continue the Loader run.
3 Write how you fixed the problem in the space provided below.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
4 What error message did you encounter next?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
5 Fix the problem and continue the Loader run.
6 Write how you fixed the problem in the space provided below.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
What error message did you encounter?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
BD FACS Loader Option
337452 Rev. A
283
Fix the problem and continue the Loader run.
7 Write how you fixed the problem in the space provided below.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
What else did you encounter or notice during the Loader run? Write your answer
in the space provided below.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
_____________________________________________________________________
_____________________________________________________________________
What is the proper way to remove the Loader cover during a run? Write your
answer in the space provided below.
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Refer to the BD FACS Loader User’s Guide for additional information about rerunning
samples and interrupting the Loader during a run.
284
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
FACS Loader
BD WorklistManager software
Acquisition software
(BD CellQuest Pro
or BD Multiset software)
Flow
Cytometer
BD LoaderManager software
337452 Rev. A
BD FACS Loader
1
1
Reminders:
• Enter current Lot IDs
(eg, TruCOUNT
beads/pellet) in
BD MultiSET software.
• Template Schedule
document comes with
each software.
• Schedule documents
do not have to be
saved in a specific
folder.
Schedule document information
used by WorklistManager:
• Preferences: export,
printing, panels, events,
banner, etc.
• Data source
• Time to view reports
• Test preferences
information
Create a Schedule
document.
337452 Rev. A
A
Preparation
• Launch BD FACSComp software.
• Use the run Lyse/No Wash Assay
selection
Perform QC appropriate
for application.
Template schedule document
1
Instrument QC
3
2
•
•
Quit BD FACSComp software.
Use the Optimization
view in BD FACSComp software or
optimize in BD MultiSET software.
Save optimized settings in the
Instrument Settings Files folder.
Optimize instrument
settings using a cellular sample.
Optimization
(With BD MultiSET Software)
5
4
- Assay Settings:
Link each panel to
optimized instrument
settings. Specify Mix
Settings.
Worklist
- Enter sample info.
- Choose panel for each
sample.
- Assign rack.
Launch the software.
Sign In
In the Set Up view
choose the following:
- File Name Prefix
- Summary Report
Sample Label
- Save a Worklist
(optional)
- For each assay, select the
Locations for each report
or export file to save and
choose a Schedule
document template.
•
•
•
•
Install rack.
Install cover.
Press Run button
on cytometer.
Click Run Tests in
software.
Acquire samples.
•
•
•
•
Enter information in
BD WorklistManager software
views
Acquisition
BD FACS Loader Option Quick Reference
Save documents
in CellQuest
Experiments Folder.
• Make
Acquisition->Analysis
or Acquisition plots.
• Choose Event Count or
Time in Acquisition &
Storage and specify count
and time.
• Select a panel in the
in the Acquisition Browser.
Reminders:
Create an acquisition
document for EACH panel
you are going to use.
337452 Rev. A
B
A
Preparation
1
Perform QC
appropriate for
application.
• Launch BD FACSComp software.
• Choose the appropriate Assay
Selection.
Instrument QC
3
2
Quit BD FACSComp software.
Optimize instrument
settings using cellular
sample.
• Use the Optimization view
in BD FACSComp software or
use BD CellQuest Pro
software.
• Save optimized settings in the
Instrument Settings Files folder.
Optimization
5
4
• Install rack.
• Install cover.
• Press Run button
on cytometer.
• Click Run Tests in
software.
Acquire samples.
Enter information in
BD WorklistManager software
views.
• Launch the software.
• Sign in.
• In the Set Up view, choose
the following:
- File Name Prefix
- Summary Report
Sample Label
- To save a worklist
(optional)
- The Locations to save
Data Files and Export
Files.
- Assay Settings:
Link each CellQuest
panel with instrument
settings and specify
Mix Settings.
• Worklist view
- Enter sample info.
- Choose panel for each
sample.
- Assign rack.
Acquisition
(With BD CellQuest Pro Software, excluding DNA applications)
BD FACS Loader Option Quick Reference
Save documents
in CellQuest
Experiments Folder.
Reminders:
• Make
Acquisition->Analysis
or Acquisition plots.
• Choose Event Count or
Time in Acquisition &
Storage and specify count
and time.
• Select a panel in the
in the Acquisition Browser.
Create an acquisition
document for EACH panel
you are going to use.
337452 Rev. A
B
A
Preparation
1
Perform QC
appropriate for
application.
• Run Lyse/Wash or
Lyse/No Wash
in BD FACSComp software
• Use the DNA Experiment
Document to:
1. Check the
following with CEN
particles:
- Resolution
- Linearity
2. Check the following
with CTN particles:
- Doublet Discrimination
Instrument QC
3
2
Save optimized
settings in Instrument
Settings Files folder.
Optimize instrument
settings using cellular
sample.
Optimization
5
4
Acquire samples.
•
Install rack.
•
Install cover.
•
Press Run button
on cytometer.
•
Click Run Tests in
software.
Enter information in
BD WorklistManager software
views.
• Launch the software.
• Sign in.
• In the Set Up view,
choose the following:
- File Name Prefix
- Summary Report
Sample Label
- To save a Worklist
(optional)
- The Locations to save
Data Files and Export
Files
- Assay Settings:
Link each BD CellQuest
panel with instrument
settings and specify
Mix Settings.
• Worklist
- Enter sample info.
- Choose panel for each
sample.
- Assign rack.
Acquisition
(With BD CellQuest Pro Software; DNA applications)
BD FACS Loader Option Quick Reference
Appendix A
Theory
The following topics are discussed in this appendix:
Theory
•
Introduction to flow cytometry theory
•
Compensation theory
337452 Rev. A
285
286
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Introduction to Flow
Cytometry
337452 Rev. A
What Do All These Things Have in
Common?
algae
chromosomes
blood
cells
protozoa
2
1
What Can a Flow Cytometer
Tell Us About a Cell?
• Its relative size (Forward Scatter—FSC)
• Its relative granularity or internal complexity (Side
Scatter—SSC)
• Its relative fluorescence intensity
3
Properties of FSC and SSC
Right Angle Light Detector
α Cell Complexity
Incident
Light
Source
Forward Light Detector
α Cell Surface Area
Forward Scatter—diffracted light
• Related to cell surface area
• Detected along axis of incident light in the forward direction
Side Scatter—reflected and refracted light
• Related to cell granularity and complexity
• Detected at 90° to the laser beam
4
2
400
600
Neutrophils
Monocytes
200
Side Scatter
800
1000
Lysed Whole Blood
0
Lymphocytes
0
200
400
600
800
1000
Forward Light Scatter
5
Basophil
Lymphocyte
Monocyte
Platelets
Eosinophil
Red Blood Cells
Neutrophil
6
3
What is Fluorescent Light?
O
HO
λ = 488 nm
λ ≅ 520 nm
C
Incident
Light Energy
Emitted Fluorescent
Light Energy
CO2H
Fluorescein
Molecule
Antibody
• The fluorochrome absorbs energy from the laser.
• The fluorochrome releases the absorbed energy by:
− vibration and heat dissipation.
− emission of photons of a longer wavelength.
7
Emission Spectra
FITC
1000
RPE
PI
APC PerCP PerCP-Cy5.5
800
600
400
200
0
400
450
500
550
600
650
700
750
Wavelength (nm)
8
4
Fluorescence
Emitted fluorescence intensity proportional to binding sites
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
FITC
FITC
FITC
Fluorescent Intensity
9
10 3
10 2
10
1
10
0
CD19 PE
10 4
Two-color cell analysis
10 0
10 1
10 2
10 3
10 4
CD3 FITC
10
5
A Cytometer Needs
a Combined System of:
Fluidics
To introduce and focus the cells for interrogation
Optics
To generate and collect the light signals
Electronics
To convert the optical signals to proportional digital signals,
process the signals, and communicate with the computer
11
BD FACSCalibur Fluidics
Air Filter
Air Pump
Flow Cell
Sheath
Regulator
Sheath
Sheath Filter
Waste
Sample
Regulator
Sample
12
6
Sample Flow
Low Differential Pressure
Low Sample
Pressure
12µl/min
High Differential Pressure
High Sample
Pressure
60µl/min
Laminar
Flow
Laminar
Flow
Sheath
Sheath
Sample
Sheath
Sheath
Sample
13
Optics
• Excitation optics consist of:
− A laser
− Lenses to shape and focus the laser beam
• Collection optics consist of:
− A collection lens to collect light emitted from the
particle-laser beam interaction
− A system of optical mirrors and filters to route
specified wavelengths of emitted light to designated
optical detectors
14
7
Optical Filters
Longpass
460
500
Shortpass
540
460
LP 500
500
540
SP 500
Bandpass
460
500
540
BP500/50
15
BD FACSCalibur Optics
Detector
FL1
Filter
530/30nm
Color
Green
Fluorochrome
FITC
FL2
585/42nm
Yellow/Orange
PE
FL3
670nm LP
Dark Red
PerCP, PerCP-Cy5.5
FL4
661/16nm
Red
APC
16
8
Collection Optics
FL1
530/30
SSC
FL2
FL4
488/10
585/42
90/10 Beam Splitter
661/16
DM 560SP
DM 640LP
670LP
Half Mirror
Fluorescence
Collection Lens
FL3
Beam Combiner
488 nm
Blue Laser
Flow
Cell
Red Diode Laser
~635 nm
488/10
FSC Diode
Focusing
Lens
17
Review
We have:
• created an illumination region with the
excitation optics.
• passed the cells precisely through the
illumination region using hydrodynamic focusing.
• directed the emitted light signals to specific
detectors (PMTs) by the collection optics.
18
9
Electronics
• Converts analog signals to proportional digital
signals
• Analyzes voltage pulse height, area, and width
• Interfaces with the computer for data transfer
19
Laser
Voltage
Creation of a Voltage Pulse
Laser
Voltage
Time
Laser
Voltage
Time
Time
20
10
Conversion of Optical Signals to
Proportional Electronic Signals
Voltage Pulses
LIN
Signal Out
Photon
In
Voltage In
Parameter
LOG
PMT
Power Supply
SSC
Time
FL1
LOG
Time
Levels 150–999 Volts
FL2
Time
SSC
FL1
FL2
FL3
FL4
FL3
Time
FL4
Time
21
Conversion of Optical Signals to
Proportional Electronic Signals
Voltage Pulses
Photon
In
FSC
LIN
Signal Out
PrePreAmplifier
Photodiode
Time
LOG
Parameter
Levels
FSC
E00
E01
E02
E03
E-1
FACSCalibur
LIN
22
11
Linear vs Log Amplification
23
Pulse Height
Volts
Quantification of a Voltage Pulse
Pulse Area
0
Pulse
Width
Time
(µ Seconds)
24
12
Pulse Height Analysis
and Digital Conversion
Analysis of
Pulse Height
Voltage Pulses
FSC
Analog-to-Digital
Conversion
SSC
Time
Analog-to-Digital
Converter
Time
0.01 V/Channel
GPIO
Interface
FL2
FL1
Time
0
1000
FL3
Time
Channels
FL4
Time
Time
25
Dot Plot
List-Mode Data
Event 1
1000
SSC
FL1
FL2
30
60
638
840
Event 2
100
160
245
85
Event 3
300
650
160
720
1000
840
800
600
FL2
FL2
FSC
400
200
85
0
0
200
400 600
FL1
800
1000
800
600
400
200
0
0
200
245
400
600 800
1000
FL1
638
26
13
FL2
Review
Time
FL1
SSC
Time
FSC
Data
Processor
FSC
SSC
Time
FL1
Time
Time
Time
FL2
FL3
FL4
27
14
Compensation
337452 Rev. A
Emission Spectra
FL2
585/42
FL3
650 and Above
Relative Intensity
FL1
530/30
500nm
550nm
600nm
650nm
700nm
Wavelength (nm)
FITC
PE
PerCP
2
1
FITC Fluorescence Overlap
FL2
585/42
Relative Intensity
FL1
530/30
500nm
550nm
600nm
650nm
700nm
W ave leng t h ( nm)
3
FITC Compensation
585/42
FL2
550nm
600nm
Relative Intensity
FL1
530/30
500nm
650nm
700nm
W a v e le n g t h ( n m )
Figure A
Figure B
Detector - % Signal
Figure C
4
2
FITC Compensation
To
Lower
Cluster
Figure B
Increase %
Subtracted
Figure A
FL1
FL2
585/42
Relative Intensity
530/30
24.8% of the Signal
Sensed in FL2
500nm
550nm
600nm
650nm
700nm
Wave leng t h ( nm )
Figure C
5
PE Fluorescence Overlap
FL3
650 and Above
FL2
585/42
Relative Intensity
FL1
530/30
500nm
550nm
600nm
650nm
700nm
Wa v e l eng t h ( nm )
PE
6
3
PE Compensation
FL3
650 and Above
FL2
585/42
Relative Intensity
FL1
530/30
500nm
600nm
550nm
650nm
700nm
Wav e leng th ( nm)
7
PerCP Fluorescence
FL1
FL2
530/30
585/42
FL3
650 and Above
Relative Intensity
Where Would the PerCP
Population Fall?
500nm
550nm
600nm
650nm
700nm
Which Compensation
Network?
W av e le ng t h ( nm )
8
4
Compensation Examples
Optimal Compensation
Under Compensation
Not enough
subtracted
Over Compensation
Too much
subtracted
9
5
Appendix B
Worksheets
Worksheets
337452 Rev. A
287
Compensation Worksheet
Place an up or down arrow in the appropriate box to show how the compensation
should be adjusted. Additionally, indicate whether the population(s) is over- or
undercompensated in the spaces provided. If no change is necessary, leave the box and
space(s) blank. Assume you are running beads stained with the appropriate
fluorochromes. (See compensation slides in Appendix A, if needed.)
288
FL3 – %FL2
FL2 – %FL3
FL2 – %FL1
FL1 – %FL2
FL3 – %FL4
FL4 – %FL3
FL1 – %FL2
FL2 – %FL1
FL3 – %FL2
FL2 – %FL3
FL4 – %FL3
FL3 – %FL4
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Instrument Controls Exercise
1 Make sure the FSC and SSC amplifiers are in Linear mode. Adjust the appropriate
controls to place the unlabeled BD CaliBRITE beads in the center of the FCS vs
SSC plot.
2 Make sure the FL1 and FL2 amplifiers are in Log mode. Adjust the appropriate
controls to place the unlabeled BD CaliBRITE beads in the lower left corner of
the FL1 vs FL2 plot.
3 Using the mixed beads, adjust the appropriate compensation networks while
viewing the FL1 vs FL2 plot to correct for over- or undercompensation of the
beads.
Worksheets
337452 Rev. A
289
4 Change the FSC Detector to E01. Which direction do the events move?
____________________________________________________________________ .
5 Change the FSC Detector back to E00. Change the FSC Detector to E-1. Click
Pause, then Restart. What happens? Why?
_____________________________________________________________________
_____________________________________________________________________
Change the FSC Detector back to E00.
6 Change the SSC Amplifier to Log. What happens to the events?
_____________________________________________________________________
_____________________________________________________________________
Change the SSC Amplifier back to Lin.
7 Change the Threshold to FL3. Click Pause, then Restart. Why do the events
disappear?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Change the Threshold back to FSC.
8 Increase the FL1–%FL2 Compensation. Look at the PE bead population. The
population is _____________ compensated. Compensate correctly.
9 Decrease the FL2–%FL1 Compensation. Look at the FITC bead population. The
population is ______________ compensated. Compensate correctly.
10 Increase or decrease the FL1 or FL2 PMT voltage. Which Compensation settings
need to be changed as a result of the PMT change?
_____________________________________________________________________
_____________________________________________________________________
290
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Day 1 Review
If you need help answering the following questions, see the appropriate module, as
indicated in parentheses.
1 What cellular property most influences forward scatter (FSC)? (Intro to Flow,
Appendix A)
_____________________________________________________________________
2 What cellular property most influences side scatter (SSC)? (Intro to Flow,
Appendix A)
_____________________________________________________________________
3 Why is it necessary to turn on the computer after the cytometer?
(BD FACSCalibur System)
_____________________________________________________________________
_____________________________________________________________________
4 Describe the daily shutdown and monthly maintenance procedures recommended
for proper maintenance of the cytometer. (BD FACSCalibur System)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
5 List the four functions FACSComp performs in the order they are performed.
(BD FACSComp Software)
_____________________________________________________________________
_____________________________________________________________________
6 Why is it important to add APC beads to tube A if you are running a 4-color
experiment? (BD FACSComp Software)
_____________________________________________________________________
_____________________________________________________________________
Worksheets
337452 Rev. A
291
7 Which assay selection in the Setup view would you choose if your experiments
involve a mouse cell line? (BD FACSComp Software)
_____________________________________________________________________
8 Why would the cytometer status remain in STANDBY when a tube has been
placed on the SIP and the instrument has been put in RUN mode. (BD
FACSCalibur System)
_____________________________________________________________________
_____________________________________________________________________
9 What wavelengths pass through a 650 LP filter? (Compensation Theory,
Appendix A)
_____________________________________________________________________
10 Why is compensation necessary? (Intro to Flow, Appendix A; BD CellQuest Pro
Software)
_____________________________________________________________________
11 Which compensation control needs to be adjusted? (Assume that there are red,
green, and unlabeled beads in the sample. (BD FACSComp Software)
_____________________________________________________________________
292
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
12 What instrument adjustment would you make to move the population in the
histogram plot to the left? (Instrument Controls Exercise, Appendix B)
_____________________________________________________________________
13 BD FACSComp software sets up the cytometer for all applications.
(BD FACSComp Software)
T
F
14 BD FACSComp software PMT settings should be performed manually if the event
rate is below 400. (BD FACSComp Software)
T
F
15 To display very large size cells on a FSC plot, you will most likely use the E01 FSC
detector setting. (Instrument Controls Exercise, Appendix B)
T
F
T
F
T
F
16 Data will be processed only on events above the threshold setting.
(Instrument Controls Exercise, Appendix B)
17 For best detection of dim signals, log amplification is preferred.
(Instrument Controls Exercise, Appendix B)
18 It is best to leave a tube filled with 4 mL of distilled water on the SIP.
(BD FACSCalibur System)
T
F
19 If the DCM arm is not placed under the sample tube, your sample will be directly
aspirated into the waste tank. (BD FACSCalibur System)
T
F
20 Double-clicking an alias folder opens the original. (BD FACStation Data
Management)
Worksheets
T
F
337452 Rev. A
293
Day 2 Review
See Module 4 BD CellQuest Pro Software if you need help answering the following
questions.
1 For each view, select the correct description.
View A
View B
View C
_____ Deselected
_____ Selected
_____ Active
What can you do with a plot when it is selected?
_____________________________________________________________________
_____________________________________________________________________
2 List the order in which the following settings should be optimized.
_____ a. FSC threshold level
_____ b. FL1, FL2, FL3 detectors
_____ c. Compensation networks
_____ d. FSC amplifier and SSC detector
_____ e. Gate population of interest
294
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
3 Which tubes can be used for optimizing compensation?
a. A tube containing only one positive population.
b. A tube containing a double-positive population.
c. A tube containing two mutually exclusive positive populations.
d. a and b
e. a and c
f. All of the above
4 When optimizing the instrument, why is it necessary to set a gate around the
population of interest before adjusting the detector voltages?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
5 How do you assure that no data will be stored while optimizing the cytometer?
_____________________________________________________________________
6 How do you permanently delete a region from an Experiment document?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
7 How do you make a document a stationery pad?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
8 Data files are saved as part of the Experiment document.
Worksheets
T
F
337452 Rev. A
295
9 On an instrument that has been optimized, changing the detector voltages will
necessitate a readjustment of compensation.
T
F
10 To display acquired data in a plot, you should choose File > Open Document.
T
F
FL4 Option
Answer the following questions only if you acquired 4-color samples.
1 BD FACSComp software sets FL4 PMT voltage using APC beads instead of
unlabeled beads. Why?
_____________________________________________________________________
_____________________________________________________________________
2 Explain the purpose of time-delay calibration.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
3 Why is it necessary to make sure your sheath tank cap is tightly secured when
running 4-color samples?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
296
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Day 3 Review
DNA
Answer the following questions if you completed the DNA module.
1 CEN particles in the BD DNA QC kit are used to check instrument
________________________________ and _________________________________.
CTN particles in the BD DNA QC kit are used to check
____________________________________________________________________ .
2 What particle in the DNA QC kit can be used to check laser alignment if
resolution (CV) is > 3% and linearity values are not within 1.95 – 2.05 range?
_____________________________________________________________________
3 Instrument settings need to be optimized before you acquire DNA data. What
optimization steps did you perform to optimize instrument settings for the DNA
samples acquired in class?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
4 What flow rate should you use to acquire DNA data? Why?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
5 What is the four-pass DNA analysis process recommended by Verity, the creators
of ModFit LT software?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Worksheets
337452 Rev. A
297
6 When performing an analysis on a single cycling population, regardless of the
ploidy of the sample, ModFit LT software will always report the ploidy of the
sample as diploid.
T
F
Sorting
Answer the following questions if you completed the Sorting module.
1 Name the three sort modes.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
2 Which mode yields the best purity AND recovery?
_____________________________________________________________________
3 Which flow rate should be used during sorting?
a. HI
b. MED
c. LO
4 The event rate that yields the maximum sort rate is:
a. 500-1000 events/sec
b. 1500-2000 events/sec
c. 3000-4000 events/sec
5 If the event rate is too low, what should you do to increase it?
a. Switch to a higher flow rate.
b. Concentrate your initial sample by centrifuging it and putting it in a smaller
volume of fluid.
c. Add sterile PBS to your initial sample.
d. a and b
298
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
6 The maximum number of catcher tube movements is:
a. 100 movements/sec
b. 200 movements/sec
c. 300 movements/sec
d. 400 movements/sec
7 Why would you use the Recovery sort mode?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
8 Name three ways to end a sort.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
9 Why do we recommend precoating the collection tubes with BSA?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
10 For sorting viable cells, we recommend using _______________ for sheath fluid.
11 Describe the procedure for cleaning the sort lines after a sort.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
12 You should run bleach and distilled water through the sort lines after sorting
biohazardous material.
Worksheets
T
F
337452 Rev. A
299
BD MultiSET Software
Answer the following questions if you completed the Sorting module.
1 What instrument adjustments are performed when optimizing samples stained
with BD TriTEST or BD MultiTEST reagents?
_____________________________________________________________________
2 Why is it necessary to leave a small amount of debris when optimizing the FL3
threshold?
_____________________________________________________________________
_____________________________________________________________________
3 BD Multi-Check control can be used as a process control.
T
F
4 When using BD TruCOUNT tubes, you must enter information in the WBC and
Lymph fields to calculate absolute counts.
T
F
T
F
5 Changes made to attractors during analysis can be saved.
300
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Final Review
1 Cell A is 10 microns and is not very complex. Cell B is 30 microns and is
complex. Draw a circle to represent cell A and a square to represent cell B in the
following plot. (Introduction to Flow Cytometry, Appendix A)
SSC
FSC
2 You place a tube on the cytometer and press the RUN button. The button remains
orange. What does this indicate? What would you do to troubleshoot the
problem? (BD FACSCalibur System)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
3 Why is it necessary to place a tube of DI water on the SIP rather than leaving no
tube on the SIP at the end of the day? (BD FACSCalibur System)
_____________________________________________________________________
4 In what folder are the FACSComp Calib File and Calib File.LNW stored?
(BD FACSComp Software)
_____________________________________________________________________
5 What is the purpose of time delay calibration? (BD FACSComp Software)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Worksheets
337452 Rev. A
301
6 Why is it important to input the correct lot ID information in BD FACSComp
software? (BD FACSComp Software)
_____________________________________________________________________
7 You review the Summary Report generated by BD FACSComp software and
notice FL1 Sensitivity Test failed. How would you troubleshoot this problem?
(BD FACSComp Software)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
8 If you are running samples stained with FITC and APC reagents, would you need
to adjust compensation? If yes, which compensation networks do you need to
adjust? (Compensation Theory, Appendix A)
_____________________________________________________________________
9 You created a template file that you want to protect from being accidentally
overwritten or deleted. What can you do to protect the file? (BD FACStation
Data Management)
_____________________________________________________________________
10 In the space provided, sketch how you plan on organizing your folders and files
when you return to your lab. (BD FACStation Data Management)
302
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
11 Optimization is a multistep process. List the five basic steps in the order they
should be performed. (BD CellQuest Pro Software)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
12 Which software program will monitor the daily performance of your instrument
and provide instrument settings for human lymphocyte immunophenotyping?
_____________________________________________________________________
13 You place a tube containing unlabeled and PE-labeled beads on the cytometer.
Where do you expect to see the unlabeled and PE populations in the following
plot. Assume all compensation is set at 0.0%. (Compensation Theory,
Appendix A)
FL1
FL2
14 When you increase/decrease the voltage on a PMT, you are adjusting a(n)
________________? (BD CellQuest Pro Software)
a. Detector
b. Threshold
c. Test Pulse
Worksheets
337452 Rev. A
303
15 Which tube can you use to optimize scatter? (BD CellQuest Pro Software)
a. CD3/CD8/CD45/CD4
b. Isotype control
c. CD3/CD16+CD56/CD45/CD19
d. Any of the above
16 Which type of plot displays only a single parameter? (BD CellQuest Pro
Software)
a. Histograms
b. Dot plots
c. Contour plots
d. Density plots
17 BD CellQuest Pro software gets the cytometer type and configuration from the
BDPAC (Power PC) file. Where is this file located? (Discussed in class.)
a. In the BD Files folder
b. In the Extensions folder
c. In the BD Applications folder
18 To change an FSC vs SSC plot to FL1 vs FL2 in BD CellQuest Pro software, you
would... (BD CellQuest Pro Software)
a. Go to the Edit menu
b. Go to the File menu
c. Click on the parameter labels pop-up menu on the plot
d. Go to the Stats menu
304
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
19 How do you add free text to a document in BD CellQuest software?
(BD CellQuest Pro Software)
a. Edit menu
b. File menu
c. Tool palette
d. Plots menu
20 If the cytometer is having problems communicating with the computer, which
would you check? (Demonstrated in class.)
a. BDPAC
b. GPIO cable connection
c. a and b
21 When in standby mode, the sample injection tube will drip onto the benchtop
when the support arm is to the side of the SIP. (BD FACSCalibur System)
T
F
22 For one-color acquisition, you need to set compensation. (BD CellQuest Pro
Software)
T
F
23 To define a logical gate, to which pull-down menu should you go?
(BD CellQuest Pro Software)
_____________________________________________________________________
24 Why would it not be a good idea, when acquiring data, to accept only a region
around a specific population for data storage? (Discussed in class.)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Worksheets
337452 Rev. A
305
DNA Questions
Answer the following questions only if you completed the DNA module.
1 Which pulse characteristic is best for measuring DNA content?
a. Pulse height
b. Pulse width
c. Pulse area
d. Pulse angle
2 You plan on looking at DNA cell cycle using the nucleic acid dye 7-AAD, which is
usually detected in the same parameter as PerCP. Which instrument settings
should you adjust?
a. FL1 threshold, FL1 Voltage, FL1-Width Amp Gain
b. FL2 threshold, FL2 Voltage, FL2-Width Amp Gain
c. FL3 threshold, FL3 Voltage, FL3-Width Amp Gain
3 The cleaning procedure used at shutdown should also be used immediately after
running propidium iodide or acridine orange-stained samples.
T
F
T
F
4 A G1 doublet has a greater pulse width signal than a G2 nucleus.
5 Which instrument characteristics are monitored using the DNA QC Particles Kit?
a. Sensitivity
b. Linearity
c. Compensation
d. Resolution
e. a, b, and d
f. b and d
306
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
6 Name 3 indicators that an Auto analysis of data in ModFit LT software is not
sufficient and that you need to perform a manual analysis.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
Sorting Questions
Answer the following questions only if you completed the Sorting module.
1 If you wanted to ensure that you collected the maximum possible number of a
target cell population, what sort mode could you use?
_____________________________________________________________________
_____________________________________________________________________
2 The collection tubes used for sorting should be coated with:
a. PBS
b. Nothing
c. 4% BSA
d. Paraformaldehyde
3 When would you use single cell sort mode?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
4 When sorting viable cells for culture it is advisable to:
a. Use sterile PBS for sheath fluid
b. Sterilize the instrument with 70% ethanol before sorting
c. Use sterile BSA to coat the collection tubes
d. All of the above
Worksheets
337452 Rev. A
307
5 Before sorting you should:
a. Select the LO flow rate setting
b. Define a sort region
c. Select a Sort Mode
d. Select the HI flow rate setting
e. a, b, and c
f. a, b, and d
BD FACS Loader Questions
Answer the following questions only if you completed the BD FACS Loader module.
1 In BD Worklist Manager software, if the Assay Type is CellQuest, the
BD Cellquest Pro Experiment document to be used for acquisition must be in
what folder to be found by BD Worklist Manager software?
_____________________________________________________________________
_____________________________________________________________________
2 Why is it beneficial to use Event Count or Time as the stop acquisition criterion in
a BD CellQuest Pro Experiment document used with WorklistManager software?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
3 When making a panel in a BD CellQuest Pro Experiment document to be used in
WorklistManager software, why is it a good idea to change the tube names from
the default names (Tube #)?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
308
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
4 What is an advantage of optimizing the instrument settings using the
Optimization view in BD FACSComp software?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
5 You can run BD LoaderManager software maintenance and diagnostic
procedures while in BD WorklistManager software.
T
F
6 BD FACSComp software can be launched from within BD WorklistManager
software.
T
F
7 When running more than one rack, bleach and water must be in positions 39 and
40 on each rack.
T
F
8 You can add samples to a rack that’s already been assigned if first you release the
rack.
T
F
9 To use an instrument settings file in BD Worklist Manager software it must be in
the BD Files:Instrument Settings File folder.
T
F
T
F
10 Tubes from the same panel can be split between two racks.
BD MultiSET Software Questions
Answer the following questions only if you completed the BD MultiSET Software
module.
1 What is the purpose of adjusting FL3 threshold?
_____________________________________________________________________
Worksheets
337452 Rev. A
309
2 Why was FL3 threshold rather than FSC threshold adjusted?
_____________________________________________________________________
_____________________________________________________________________
3 What is the purpose of the BD Multi-Check control product?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
4 You should always visually inspect Laboratory Reports to determine if any QC
messages appeared.
T
F
T
F
5 Changes made to attractors during analysis can be saved.
310
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Troubleshooting Exercise
For this exercise, do the following:
•
Do not use Calib settings or copy them from the printout.
•
In the table provided on page 306, list all the problems and solutions you
encounter throughout the exercise.
1 Adjust the appropriate instrument settings to place the unlabeled BD CaliBRITE
beads in the center of the FCS vs SSC plot (Figure A-1).
2 Adjust the appropriate instrument settings to place the unlabeled BD CaliBRITE
beads in the lower left corner of the FL1 vs FL2 plot (Figure A-2) and the FL3 vs
FL2 plot (Figure A-3).
FSC
FL1
FL3
Figure A-1
Figure A-2
Figure A-3
3 Using the mixed beads, adjust the appropriate compensation networks to correct
for over- or under compensation of the beads (Figures A-4 and A-5).
Worksheets
FL1
FL3
Figure A-4
Figure A-5
337452 Rev. A
311
Problems
312
Solutions
BD FACSCalibur • BD FACSort • BD FACScan Operator Course Workbook 337452 Rev. A
Download PDF
Similar pages