BD CellQuest Pro Software User`s Guide

BD CellQuest Pro Software User`s Guide
BD CellQuest Pro
Software
User’s Guide
http://www.bdbiosciences.com/
Part No. 341753 Rev. A
November 2000
BD Biosciences
2350 Qume Drive
San Jose, CA 95131-1807
USA
Tel (800) 448-2347
Fax (408) 954-2347
Ordering Information
Tel (800) 223-8226
Asia Pacific
Becton Dickinson and Company
Tel (65) 860-1478
Fax (65) 860-1590
Brazil
Becton Dickinson Ind. Cirúrgicas Ltda.
Tel (55) 11 5185-9833
Fax (55) 11 5185-8644
Canada
Becton Dickinson Canada, Inc.
Tel (905) 822-4820
Fax (905) 855-1243
Europe
Becton Dickinson European HQ
Tel (32) 53-720211
Fax (32) 53-720450
Japan
Nippon Becton Dickinson Company, Ltd.
Tel (81) 3-5413-8251
Fax (681) 3-5413-8155
Mexico
Becton Dickinson Mexico S.A. De C.V.
Tel (525) 237-1200
Fax (525) 237-1288
BD Biosciences • 2350 Qume Drive • San Jose, California • 95131-1807
© 2000, 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.
CellQuest Pro software © 2000, Becton, Dickinson and Company. This software is the property of Becton, Dickinson and
Company. Each sale of a stored unit of this software grants the purchaser a nontransferable, nonexclusive, personal license.
This software may not be duplicated, reproduced, or copied in any form or by any means whatsoever, except as otherwise
permitted by law.
This program was written with MacApp®, © Apple® Computer, Inc. The MacApp software is proprietary to Apple
Computer, Inc. and is licensed to BD Biosciences for distribution only for use in combination with this software.
Apple Computer, Inc. makes no warranties whatsoever, either expressed or implied, regarding this product, including
warranties with respect to its merchantability or its fitness for any particular purpose.
Apple, the Apple logo, Mac, Macintosh, Power Macintosh, and QuickTime are trademarks of Apple Computer, Inc.,
registered in the U.S. and other countries. Finder is a trademark of Apple Computer, Inc.
Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated.
CaliBRITE, CellQuest, CHRONYS, CONSORT, FACS, FACSCalibur, FACScan, FACSComp, FACSConvert,
FACSNet, FACSort, FACStarPLUS, FACStation, FACS Vantage, LYSYS, MultiSET, ProCOUNT, and SimulSET are
trademarks of Becton, Dickinson and Company.
All other company and product names may be trademarks of the respective companies with which they are associated.
Guide written by Kim Gautho; edited by Joan Miller; produced by Pushpa MacFarlane.
History
Revision
Date
History
A
11/00
New software features
Contents
About This Guide
ix
Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
x
Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xi
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xi
Chapter 1: Introduction
13
Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
Compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
Other Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
Installing CellQuest Pro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17
Installing BD Inits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
19
Launching CellQuest Pro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
Registering the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
Quitting CellQuest Pro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
Chapter 2: Becoming Familiar with CellQuest Pro Software
23
CellQuest Pro Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
Default File Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
Experiment Document Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26
Tool Palette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28
View Area Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30
iii
Inspector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
Changing Text Style and Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
32
Changing Object Geometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35
Customizing Experiment Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
Document Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
37
Text Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
40
Color Palette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
42
Banners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
Grids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
45
Chapter 3: CellQuest Pro Plots
iv
49
Creating Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
52
Deleting Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
55
Formatting Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56
Dot Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
56
Contour Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
58
Density Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
60
3D Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
Plot Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
63
Formatting Histograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
65
Creating Overlaid Histograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66
Using Histogram Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
70
Saving Experiment Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
Saving Experiment Documents as Templates . . . . . . . . . . . . . . . . . . . . . . . . . . .
73
Saving Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
75
BD CellQuest Pro Software User’s Guide
Chapter 4: Optimizing Instrument Electronics
77
Performing Instrument Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
Instrument Controls in CellQuest Pro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
79
Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
80
Amplifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
82
Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
83
Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84
Optimizing the Instrument for Biological Samples . . . . . . . . . . . . . . . . . . . . . . . . . .
85
Starting Up the Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
86
Setting Up an Acquisition Experiment Document . . . . . . . . . . . . . . . . . . . . . . .
86
Restoring Calib Settings to the Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
88
Adjusting FSC, SSC, and FSC Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
90
Gating on the Population of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
91
Adjusting the Fluorescence Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
93
Adjusting Fluorescence Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96
Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
99
Saving Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100
Restoring Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100
Printing Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
101
Checking Cytometer Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
101
Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
105
Using the FACSCalibur or the FACSort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
105
Using the FACSVantage SE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
106
Chapter 5: Acquiring Data
111
Using the Acquisition View of the Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
112
Getting Ready to Acquire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
115
Defining Global Acquisition & Storage Settings . . . . . . . . . . . . . . . . . . . . . . . .
115
Defining File Name and Storage Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
122
Defining Parameter Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
124
Contents
v
Setting Up an Acquisition Tube List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
Editing Tubes Using the Inspector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
126
Creating Plots and Associating With Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
127
Defining Tube-Specific Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
128
Using Counters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
Displaying Live Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
132
Acquiring Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
133
Monitoring Acquisition in the Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
135
Pausing Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
136
Ending Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
136
Optional Acquisition Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
137
Using the Reagent List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
137
Creating Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
138
Defining Custom Keywords . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
141
Chapter 6: Regions and Gates
vi
143
Creating Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
144
Histogram Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
144
Rectangular, Elliptical, and Polygonal Regions . . . . . . . . . . . . . . . . . . . . . . . . . .
145
Snap-To Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
148
Editing Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
150
Rotating Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
151
Deleting or Hiding Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
152
Using the Region List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
154
Copying Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
155
Defining Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
157
Using Combined Regions in Gate Definitions . . . . . . . . . . . . . . . . . . . . . . . . . .
157
Using the Gate List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
159
BD CellQuest Pro Software User’s Guide
Using Multicolor Gates for Dot Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
160
Setting Up a Gate List for Multicolor Gating . . . . . . . . . . . . . . . . . . . . . . . . . . .
162
Highlighting Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
162
Chapter 7: Statistics
165
Calculating Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
166
Automatic vs Manual Calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
167
Selecting Log Data Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
168
Calculating Percent Gated and Percent Total Events . . . . . . . . . . . . . . . . . . . . .
169
Calculating Means . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
170
Using Histogram Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
Creating Histogram Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
Displaying a Marker List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
172
Copying Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
Converting Markers to Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
174
Displaying Histogram Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
175
Generating Overlaid Histogram Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
176
Calculating Percentages From Overlaid Histograms . . . . . . . . . . . . . . . . . . . . . .
176
Calculating Kolmogorov-Smirnov (K-S) Statistics . . . . . . . . . . . . . . . . . . . . . . .
179
Creating Quadrant Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
181
Editing Quadrant Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
181
Displaying Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
182
Editing Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
184
Formatting Statistics Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
190
Using the Expression Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
191
Editing Expressions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
194
Chapter 8: Analyzing Data
195
Using the Analysis Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contents
196
vii
Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
198
Setting Up an Analysis Experiment Document . . . . . . . . . . . . . . . . . . . . . . . . . .
199
Displaying Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
202
Using the Next Data File Command . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
204
Analyzing Groups of Data Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208
Using the Load Sample Feature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208
Setting Up Batch Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
213
Saving FCS Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
215
Exporting Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
216
Using Quantitative Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
217
Setting Up for Quantitative Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
218
Chapter 9: Publishing and Presenting Data
225
Exporting Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
226
Helpful Hints for Publication-Quality Graphics . . . . . . . . . . . . . . . . . . . . . . . . .
227
Saving Experiment Documents as PDFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
228
Creating a PDFWriter Shortcut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
229
Glossary
231
Appendix A: Keyboard Shortcuts
233
Index
237
viii
BD CellQuest Pro Software User’s Guide
About This Guide
This user’s guide describes the setup and operation of BD CellQuest™ Pro
software. This guide is intended for laboratory personnel and flow cytometry
operators experienced in flow cytometry instrument operation. For information on
using the FACS™ Loader or other instrument components, refer to the
appropriate user’s guide.
The CellQuest Pro Software User’s Guide assumes you have a working knowledge of
basic Macintosh® operation. If you are not familiar with the Macintosh computer,
refer to the appropriate user’s guide provided by Apple Computer, Inc.
Before using CellQuest Pro software, review the ReadMe file that was copied to
your hard disk during software installation. The file contains late-breaking
information that is not printed in this user’s guide.
First-time users of CellQuest Pro software should read Chapter 1 to learn about
basic software functions and compatibility, Chapters 2 and 3 to learn about
CellQuest Pro components, and Chapters 6 and 7 to learn about analysis tools like
regions, gates, and statistics. You will find instructions for routine acquisition and
analysis in Chapter 4, Chapter 5, and Chapter 8. For tips on publishing and
presenting data, see Chapter 9. Appendix A has a useful list of keyboard shortcuts.
ix
Conventions
The following tables list conventions used throughout this guide.
Table 1 Notice icons
Icon
Notice Type
Description
NOTE
Describes important features or instructions.
m
CAUTION
Alerts you to potential loss of data or potential damage to
an application, system, or device
H
WARNING
Alerts you to potential personal injury
Table 2 Text conventions
x
Convention
Description
Italics
New or unfamiliar terms are listed in italics on their first
appearance in the text. A glossary of terms is listed at the end
of this manual.
z
This is the Command key symbol. Keyboard shortcuts use the
Command key in combination with another indicated
keystroke (eg, z-K means press Command and K
simultaneously).
BD CellQuest Pro Software User’s Guide
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.
• Contact your local BD Biosciences service representative if you need
additional assistance.
When you contact BD Biosciences, have the following information
available:
•
product name, part number, and serial number
•
any error messages
•
details of recent system performance
For technical assistance from within the US, call:
(800) 448-2347, prompt #1
For assistance from within Canada, call:
(800) 268-5430
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.
About This Guide
xi
xii
BD CellQuest Pro Software User’s Guide
1
Introduction
• requirements for running CellQuest Pro
• compatibility with other programs
• installation of CellQuest Pro software
• instructions for launching and quitting CellQuest Pro software
13
CellQuest Pro software allows you to acquire and analyze data from your flow
cytometer on a Macintosh computer. Working in the CellQuest Pro Experiment
document window, you can create several types of plots, including multicolor
contour plots and overlaid histograms, and you can generate statistics for dot plots,
histograms, density plots, 3D plots, and contour plots. The versatile graphics tool
palette helps you produce presentation-quality documents. You can also save
everything in an Experiment document, including plots, regions, gates, markers,
statistics, calculated expressions, Browser contents, annotations, and color
preferences, and then restore it all later.
Requirements
Hardware
• BD FACSCalibur™, FACScan™, FACSort™, or BD LSR flow cytometer;
FACS Vantage™ family of cytometers (FACS Vantage, FACSVantage SE)
• FACStation™ computer (Power Macintosh® blue and white G3, or G4
purchased through BD Biosciences)
Other platforms might also be supported; contact your BD representative
for detailed information.
m CAUTION
If you have a modem on your system, BD recommends turning it
off while running our software.
• Universal Serial Bus™ (USB) security module
NOTE The security module is provided with the software.
• (optional) FACS™ Loader for acquisition; LoaderManager software
version 3.2 or greater is required.
14
BD CellQuest Pro Software User’s Guide
Software
• Mac® OS 8.6 and 9.0.4
•
file sharing and program linking turned off
•
virtual memory on
Other versions of the system software might also be supported; contact your
BD representative for detailed information.
• BD Inits version 3.4 or greater (see Installing BD Inits on page 19)
• Apple® QuickTime™ version 4.0 or greater
• LoaderManager software version 3.2 or greater, for
cytometers equipped with the FACS Loader
BD Biosciences has tested many common INITs (eg, extensions, control panels)
that do not adversely affect software performance; however, some INITs might
interfere with the software. Therefore, we do not recommend installing additional
INITs.
Compatibility
Data Files
CellQuest Pro reads and writes FCS 2.0 data files. See Other Software on page 16.
HP files must be moved to the Macintosh using a network transfer product such as
FACSNet™ Mac or CONSORT™ File Exchange; then they must be converted
to FCS 2.0 format using FACSConvert™ software. Refer to the appropriate
documentation for instructions.
CellQuest Pro analyzes data files produced by CONSORT 30, FACScan Research,
FACStarPLUS™ Research, LYSYS™ II, SimulSET™, MultiSET™,
ProCOUNT™, or any other BD software except CHRONYS™.
Chapter 1: Introduction
15
Other Software
CellQuest
• CellQuest Pro can open and read data files and Experiment
documents created with CellQuest version 3.3 or greater;
however, CellQuest Pro Experiment documents cannot be read
by CellQuest.
• Preferences from CellQuest version 3.3 or greater are not deleted
or overwritten by CellQuest Pro. CellQuest Pro preferences will
retain older version CellQuest preferences.
16
CloneCyt™ Plus
For single-cell sorting, CellQuest Pro, in conjunction with
CloneCyt Plus software and a FACSVantage SE flow cytometer,
helps identify a single cell’s position on a plot when the
corresponding clone number is known.
WorklistManager
CellQuest Pro Experiment documents can serve as assay templates
in WorklistManager.
Adobe® Acrobat®
software
With the addition of the Adobe PDFWriter, CellQuest Pro
Experiment documents can be saved as Adobe PDF (portable
document format) files that can be viewed on any computer using
Adobe’s free Acrobat Reader software.
BD CellQuest Pro Software User’s Guide
Installing CellQuest Pro
Follow these steps to install CellQuest Pro software on your computer’s hard disk.
1 Shut down the computer.
2 Attach the security module which is packaged with this user’s guide.
CellQuest Pro will not launch if the security module is not attached.
•
blue and white Power Macintosh G3 computer—plug the USB security
module into the USB port on the keyboard.
•
Power Macintosh G4 computer—plug the USB security module into
the USB hub.
For help in connecting the security module to other FACStation computers,
contact the BD Customer Support Center.
NOTE The security module does not need to be connected at both ends.
3 Restart the computer.
4 Insert the FACStation Basic Software CD-ROM disc into the CD-ROM
drive.
5 Double-click the CD-ROM icon.
A window appears showing the contents of the CD-ROM disc. The
FACStation Basic Software disc includes installers for several BD
applications, electronic documentation, and Adobe Acrobat Reader, used to
open and navigate through the electronic documentation. Review the
FACStation ReadMe file for more information.
6 Double-click the Install CellQuest Pro icon.
Chapter 1: Introduction
17
7 Follow the instructions in the dialog box to finish installing the software.
NOTE When prompted, click the Switch Disk button as needed to select a
destination disk.
The installer loads CellQuest Pro and its support files on the selected hard
disk. It also removes any existing version of CellQuest Pro provided you have
not renamed the application or moved it from the CellQuest Pro folder. A
message appears when installation is complete.
8 Click Quit or Restart when you see a message reporting a successful
installation.
You must restart your computer to detect the Eve Init the installer places in
the Extensions Folder in the System Folder. This Init is required to run
CellQuest Pro on a Power PC computer.
The CellQuest Pro installer adds its files to the BD Applications folder and
the BD Files folder (Figure 1-1). The CellQuest Pro folder, located in the
BD Applications folder, contains the CellQuest Pro application, a copy of
the ReadMe file, and a Sample Files folder. The CellQuest Pro Files folder,
located in the BD Files folder, is the default storage location for Experiment
documents.
The installer also creates a CellQuest Pro alias under the Apple menu.
18
BD CellQuest Pro Software User’s Guide
Figure 1-1 BD Applications and BD Files folder contents
Installing BD Inits
All BD applications that acquire data from a flow cytometer require BD startup
files, or BD Inits, that extend the capabilities of the computer. These files are
already installed in all BD FACStation computer systems. However, if it is
necessary to reinstall or update BD Inits, follow these instructions.
1 Insert the FACStation Basic Software CD-ROM disc into the CD-ROM
drive.
2 Double-click the CD-ROM icon.
3 Double-click the Install BD Inits icon.
4 Follow the instructions that appear on the screen.
Chapter 1: Introduction
19
5 Click Restart when installation is complete.
The installer automatically places the following BD Init files into the
Macintosh System Folder:
•
Acquisition Library (AcqLibPPC) and the BDPACDriver into the
Extensions folder
•
BDPAC into the Control Panels folder
•
BDPACInit into the Startup items folder
Launching CellQuest Pro
Before launching the software, verify that Apple QuickTime version 4.0 or greater
is installed on your system and that the security module is attached. CellQuest Pro
will not start without the security module attached.
Open the CellQuest Pro application one of several ways:
• Choose CellQuest Pro from the Apple menu.
CellQuest Pro opens with a new, untitled
Experiment document.
• Double-click the CellQuest Pro application icon
in the CellQuest Pro Folder on your hard disk.
CellQuest Pro opens with a new, untitled
Experiment document.
• Double-click a saved CellQuest Pro Experiment
document. CellQuest Pro opens to the saved
Experiment document.
• Double-click a saved CellQuest Pro FCS data file
or exported statistics file. CellQuest Pro launches,
but neither the file nor the statistics is opened.
You must open an Experiment document to use
the program.
20
BD CellQuest Pro Software User’s Guide
application icon
Experiment
document icon
FCS data
file icon
exported
stats file icon
• Drag one or more CellQuest Pro Experiment documents onto the
CellQuest Pro application icon. This launches CellQuest Pro and allows you
to open several Experiment documents at one time.
Registering the Software
The first time you launch CellQuest Pro software, a Registration dialog box
appears.
1 Enter your name and the name of your institution.
2 Click Save.
Quitting CellQuest Pro
Do either of the following to quit CellQuest Pro:
• Choose Quit from the File menu.
• Hold down the Command (z) key while you press the Q key (z-Q).
Chapter 1: Introduction
21
22
BD CellQuest Pro Software User’s Guide
2
Becoming Familiar with
CellQuest Pro Software
• CellQuest Pro files
• Experiment document components
• Inspector
• Experiment document customization
23
CellQuest Pro Files
This section describes the several types of files CellQuest Pro can create:
Experiment document files, FCS data files, statistical files for export, and
instrument settings files.
See page 25 for a summary of default file-naming conventions and storage
locations.
Experiment Document Files
A CellQuest Pro Experiment document is a document that can
contain user-defined plots, regions, gates, markers, statistics, tube
lists in the Browser, calculated expressions, text annotations, and
color preferences. When saving an Experiment document,
CellQuest Pro saves a reference to the data files used in the
Experiment document, not the data files themselves.
See Experiment Document Components on page 26 for general
information about Experiment documents; see page 73 for
information about saving Experiment documents.
Data Files
Flow cytometric data is stored according to a standard format, the
Flow Cytometry Standard (FCS) format, developed by the
International Society for Analytical Cytology.a After acquisition, the
parameter measurements for each event and the instrument settings
of the cytometer are automatically saved in an FCS data file.
See Saving Data on page 75 for more information about data files.
a. For a detailed description of FCS 2.0, refer to Data file standard for flow cytometry. Data File Standards
Committee of the International Society for Analytical Cytology. Cytometry. 1990;11(3):323-332.
24
BD CellQuest Pro Software User’s Guide
Statistics Files
Any combination of histogram, region, gate, and quadrant statistics
can be exported as tab-delimited text to a file, with the exception of
statistics from overlaid histograms and the Kolmogorov-Smirnov
(K-S) two-sample test. These export files can be used to transfer data
to other applications, such as spreadsheet programs or databases.
See page 216 for information about exporting statistics.
Instrument Settings Files
An instrument settings file contains detectors/amps settings, the
threshold parameter and value, compensation settings, pulse
processing settings (if applicable to your cytometer), and special
setup values (if applicable to your cytometer). These settings are
saved automatically in the text section of FCS files; they can also be
saved in a separate instrument settings file.
See page 99 for information about saving and restoring instrument
settings.
Default File Naming
Table 2-1 summarizes the file naming conventions for the different types of
CellQuest Pro files.
Table 2-1 CellQuest Pro file names
File Type
File Name
Experiment document
Untitledn (CellQuest Pro adds a
number to each subsequent file)
FCS data file
Data.nnn
Chapter 2: Becoming Familiar with CellQuest Pro Software
25
Table 2-1 CellQuest Pro file names (continued)
File Type
File Name
Export statistics
untitled stats
Instrument settings
INSTRSettings
Experiment Document Components
When you start CellQuest Pro software, an Experiment document window
appears. The CellQuest Pro Experiment document is where you create plots for
acquisition and analysis, show statistics, create calculated expressions, enter text,
and customize preferences. Software functions are controlled using the tool palette
and the menu bar above the window. Other Experiment document window
elements include the ribbon, view area, size box, and zoom box.
menu bar
ribbon
zoom box
tool palette
view area
size box
Figure 2-1 CellQuest Pro Experiment document window
26
BD CellQuest Pro Software User’s Guide
• menu bar—displays CellQuest Pro pull-down menus with the commands
you will need to operate the software; for a complete description of the
menus, see Appendix A.
• ribbon—shows the cursor coordinates in a plot; it is often helpful to know
the cursor coordinates when you create markers or regions.
When the cursor is on a dot, density, or contour plot, the ribbon displays the
channel number or linear value of the x-axis (X) and the y-axis (Y).
When the cursor is on a histogram, the ribbon displays the channel number
or linear value of the x-axis (X) and the event count (Y).
For overlaid histograms, the Y value is blank.
• tool palette—appears when you open an Experiment document; use the
tools in the palette to create plots, regions, markers, and text. For a complete
description, see the following section.
• view area—displays all Experiment document elements you create; for a
complete description, see View Area Objects on page 30.
• zoom box—opens the window to its maximum screen size.
• size box—resizes the window.
Chapter 2: Becoming Familiar with CellQuest Pro Software
27
Tool Palette
The tool palette appears when you launch CellQuest Pro. Use the
tools in the palette to create plots, regions, markers, text, and
calculated expressions.
• To select a tool, click once to highlight it.
• To move the tool palette to a different location, click the
shaded bar at the top of the palette and drag to the new
location.
• To remove the tool palette from the desktop, click the close
box in the upper-left corner of the palette, or choose Hide
Palette from the Windows menu.
To make the palette reappear, choose Show Palette from the
Windows menu.
Here is a summary of the tool functions.
Selection tool—selects (by clicking), moves (by dragging), or
resizes (by dragging) items in the Experiment document.
C
3D
H
D
d
Plot tools—create contour (C), 3-dimensional (3D), density
(D), histogram (H), and dot (d) plots. Select the tool, click in an
unused section of the Experiment document, and drag
diagonally until the plot outline is the desired size.
For information on creating plots, see page 52.
H
Q
Marker tools—The Histogram-Marker tool (H) creates a
marker on a histogram plot allowing you to obtain statistics on
the data. The Quadrant-Marker tool (Q) draws a quadrant on a
dot, density, or contour plot. Select the tool, click in the plot,
and drag to set the marker.
See Chapter 7 for more information on setting markers.
28
BD CellQuest Pro Software User’s Guide
Zoom tools—The Zoom-In tool (+) magnifies an area of a plot.
Select the tool, click in the plot, and drag to define the area of
the plot you want to enlarge.
The Zoom-Out tool (–) returns the plot to its original size.
Select the tool and click in the plot.
R
P
H
E
S
Region tools—create regions on specific plots.
• The Rectangular-Region tool (R) creates a rectangular region
on a dot, density, or contour plot. Select the tool, click in the
plot, and drag diagonally until the region outline is the desired
size.
• The Polygonal-Region tool (P) creates a polygonal region on a
dot, density, or contour plot. Select the tool and click in the
plot to establish the starting point (first vertex). Move the
cursor to create the next vertex and click. Continue moving
the cursor and setting vertices; click the first vertex to
complete the region.
• The Histogram-Region tool (H) creates a histogram region on
a histogram plot. These regions are created just like histogram
markers. However, histogram regions are used for gates;
markers are used for statistics.
• The Elliptical-Region tool (E) creates a circular or elliptical
region on a dot, density, or contour plot. Select the tool, click
in the plot, and drag diagonally until the region outline is the
desired size.
• The Snap-To–Region tool (S) draws a region around a distinct
cluster on a dot, density, or contour plot. Select the tool and
click the cluster on the plot. The region is drawn
automatically.
For information on creating regions, see page 144.
Chapter 2: Becoming Familiar with CellQuest Pro Software
29
Text tool—creates or edits free text anywhere in the Experiment
document. Select the tool and click to position the insertion
point; type the text and click outside the text box to complete
the entry.
For information on adding free text, see page 39. For
information on customizing the style of free text, see Changing
Text Style and Color on page 32 or Text Settings on page 40.
Arrow tool—draws arrows in the Experiment document. Select
the tool, click in the view area and drag the line out. Arrows
must be started in an empty area of the Experiment document
but they can extend into objects.
Expression Editor tool—opens a dialog box where you can
enter custom mathematical expressions (formulas). The results of
your calculated expression appear on the Experiment document
with a label you have assigned.
For more information on calculated expressions, see Using the
Expression Editor on page 191.
Calculator tool—recalculates data. This tool is enabled only if
Auto Recalculate (Gates menu) is turned off. Click the tool to
recalculate data. For more information, see Automatic vs Manual
Calculation on page 167.
View Area Objects
Plots, statistics, expressions, and free text that appear in an Experiment document
view area are collectively referred to as objects. Objects appear in three different
states: active, selected, or deselected.
• An active object has a gray border (Figure 2-2); it is the target of any action
you take. Any commands you choose affect only the active object.
For example, if you make a plot active and then choose Select All from the
Edit menu, everything within that plot, such as markers and regions,
30
BD CellQuest Pro Software User’s Guide
becomes selected. On the other hand, if no objects are active and you choose
Select All, all objects in the Experiment document become selected.
To make an object active, click anywhere inside the object except on the
frame. Only one object can be active at a time.
frame
Figure 2-2 Active object (plot)
• A selected object has a black handle on each corner (Figure 2-3); it has many
properties of an active object. However, only a selected object can be
inspected, deleted, resized, or moved.
Only one object can be active at a time, but multiple objects can be selected.
To select an object, click its frame. To select more than one object, click each
frame while holding down the Shift key or click in the view area and drag the
cursor diagonally to surround the desired objects. To select all objects,
choose Select All from the Edit menu; make sure no objects are active before
choosing Select All.
selection handles
Figure 2-3 Selected object (plot)
Chapter 2: Becoming Familiar with CellQuest Pro Software
31
• A deselected object has a clear border (Figure 2-4). To deselect an active or
selected object, click anywhere outside the object.
Figure 2-4 Deselected object (plot)
Inspector
The CellQuest Pro Inspector provides an easy-to-use interface for viewing or
modifying the attributes of a single object or set of objects in the Experiment
document. For example, you can use the Inspector to change text and color
settings, resize multiple objects to the same size, align multiple objects in an
Experiment document, select or change the file displayed in an analysis plot, or
enter or change parameter labels.
The type of Inspector varies depending on the objects selected in the view area.
Common Inspector elements are discussed in this section; for Annotation
Inspectors, see page 39. For plot Inspectors, see Formatting Plots on page 56. For
statistics Inspectors, see Formatting Statistics Views on page 190.
Changing Text Style and Color
You can change the text style of any selected object or set of objects in an
Experiment document using the Text Style options in the Inspector. When a plot
is selected, for example, text attributes apply to all text within the plot, including
the plot title and axes labels.
32
BD CellQuest Pro Software User’s Guide
1 Select one or more objects in the Experiment view area.
Select more than one object by holding down the Shift key as you click
individual objects or by clicking in the view area and holding down the
mouse button as you drag across the view.
To select all objects, choose Select All from the Edit menu.
2 Choose Show Inspector from the Windows menu (z-I).
The Inspector appears, indicating in the title bar the type of object or
number of objects selected. The contents of the Inspector vary depending on
the objects selected; however, any objects including text will show Text Style
options within the Inspector window.
Inspector contents—dot plot selected
Inspector contents—multiple objects selected
Inspector contents—nothing selected
NOTE The disclosing triangles are closed in this illustration. When the
Inspector window appears on the screen, the triangles are open, revealing the
options for each category.
3 Scroll down to the Text Style header line; if needed, click once on the
disclosing triangle to display Text Style options.
disclosing triangle
to hide/show
style options
pop-up controls
Chapter 2: Becoming Familiar with CellQuest Pro Software
33
4 Specify the text style.
•
Click once on the Font pop-up control to change the font; select a font
from the menu that appears. All fonts installed on your computer are
available.
•
Enter the desired font size in the Size entry field or click on the pop-up
control and select a size from the menu that appears.
•
Specify the text style. You can select bold, italic, underlined, outlined, or
shadowed. Select condense (C) to compress spacing between characters;
select extend (E) to stretch out spacing. Remember that the selected style
and justification applies to the text in all selected objects.
•
Specify the justification (alignment) for any free text or text annotations.
Justification cannot be applied to the text in plots or statistics views.
•
Specify the text color. Click once on the colored square and choose from
the 24 available colors. You can change the available color selections
using the Color Palette, as described on page 42.
•
Click on the Background pop-up control to choose the background for
any free text or text annotation that overlaps an object. As shown in
Figure 2-5, a Transparent background reveals overlapping items in the
text field; an Opaque background hides overlapping items in the text
field.
transparent background
opaque background
Figure 2-5 Text background options
5 Close the Inspector by clicking inside the close box or by choosing Hide
Inspector from the Windows menu.
34
BD CellQuest Pro Software User’s Guide
Changing Object Geometry
You can resize multiple objects to the same size or align multiple objects in an
Experiment document using the Geometry feature. Location and size settings are
in inches or centimeters. These settings are specified in the Grid Settings dialog
box, described on page 45.
1 Select one or more objects in the Experiment view area.
For example, to make a series of plots all the same size, select all the plots in
the Experiment view area.
2 Choose Show Inspector from the Windows menu (z-I).
3 Scroll down to the Geometry header line; if needed, click once on the
disclosing triangle to display Geometry options.
disclosing triangle
to hide/show
geometry options
4 Enter a location and size of the selected objects.
You can change unit settings from inches to centimeters in the Grid dialog
box (see page 45). If two or more items are selected, only attributes that are
common to all the selected objects are displayed.
•
To make all selected objects the same size, enter a width and height in
the Size boxes.
•
To align multiple objects horizontally, enter a distance from the top of
the page; leave the Left distance blank.
•
To align multiple objects vertically, enter a distance from the left side of
the page; leave the Top distance blank.
Chapter 2: Becoming Familiar with CellQuest Pro Software
35
Customizing Experiment Documents
You can customize certain features of an Experiment document and save them
with the document. These include the document size, the presence and location of
text or annotations, the text and color settings for all objects in the view area, the
appearance of a custom banner, and the presence or absence of grids. These
features are described in this section.
Document Size
CellQuest Pro sets the size of each new Experiment document to a standard
8.5 x 11-inch page with 1-inch margins at the top and bottom. The view area is set
to this page size. To increase the size of the view area, you can increase the
Experiment document size up to a maximum of 20 pages.
The actual number of pages needed to print an Experiment document is
determined by the paper size selection and the reduction or enlargement
percentage set in the Page Setup dialog box. CellQuest Pro recalculates the
minimum number of pages needed to show all the information in a document
when changes are made to Page Setup. Refer to your Macintosh user’s guide for
information about setting up pages for printing.
1 Choose Document Size from the File menu.
2 Click the rectangles to the right or below the current page selection to add
additional pages (Figure 2-6).
Figure 2-6 Document Size dialog box
36
BD CellQuest Pro Software User’s Guide
Each rectangle represents one page. As you add pages, the corresponding
height and width measurements are updated to the left of the grid.
NOTE The number of pages selected appears in an uneditable field.
CellQuest Pro automatically adds pages to maintain a rectangular shape in
the grid. The number of pages cannot be reduced if there is any overlapping
information on a page you want to eliminate.
3 Specify the scale (inches/cm) and page sequence.
The page sequence refers to the printing order.
4 Click OK when you are finished.
The view area will increase to encompass the number of pages you have
selected; use the scroll bar to navigate within the enlarged view area.
Page breaks are indicated by a dotted line. Do not place Experiment
document objects on the dotted line representing the page break.
Text
There are two ways to add text to an Experiment document. The first method,
annotating, allows you to extract and display text that was saved with the FCS data
file. Annotations are linked to the data file and are updated when the data file
displayed in a plot changes. The second method, adding free text, allows you to
add any text you type or paste. You can place text and annotations anywhere in the
Experiment document.
Adding an Annotation
Use the Annotation feature to display text from an FCS data file. If a linked data
file cannot be found, the annotation changes to “***”; if a plot is deleted, its
annotation is also deleted.
1 Select a plot.
Chapter 2: Becoming Familiar with CellQuest Pro Software
37
2 Choose Annotation from the Plots menu and hold down the mouse button
to view the annotation pull-down submenu.
A submenu with a list of annotation types appears (Figure 2-7). The
available choices vary depending on which items were saved in the data file.
Figure 2-7 Annotation submenu from Plots menu
3 Choose the type of annotation you want from this list.
The annotation, drawn from the current data file, appears. When the
annotation is selected, handles appear around it.
selected annotation
4 Select the annotation if you want to edit it.
You can change the text style or background of a selected annotation using
the Inspector or the Text Settings dialog box, but you cannot edit the
annotation text. See Changing Text Style and Color on page 32 or Text
Settings on page 40.
38
BD CellQuest Pro Software User’s Guide
•
To move a selected annotation to a different location, click and drag it.
Annotations can be placed anywhere in the Experiment document.
•
To change the width of a selected annotation, drag a handle in or out.
•
To delete a selected annotation, press Delete or choose Cut or Clear
from the Edit menu.
•
To change the type of annotation, choose Show Inspector from the
Windows menu (z-I) and choose a different annotation from the
Attribute pop-up menu.
Adding Free Text
1 Select the Text tool from the tool palette (
).
2 Click on the Experiment document to position the insertion point.
You can place free text anywhere in the Experiment document. A blinking
vertical bar appears at the insertion point.
3 Enter the text you want.
The text wraps to the next line after reaching a default width. Click
anywhere outside the text box to complete the entry.
4 Select the annotation to move or delete it.
When the free text is selected, handles appear around it.
selected free text
Chapter 2: Becoming Familiar with CellQuest Pro Software
39
•
To move selected free text to a different location, click and drag it.
•
To change the width of selected free text, drag a handle in or out.
•
To delete selected free text, press Delete or choose Cut or Clear from the
Edit menu.
5 To edit free text, select the Text tool and position the cursor where you want
to edit the text.
You can also change the text style or background of selected free text using
the Inspector or the Text Settings dialog box. See Changing Text Style and
Color on page 32 or Text Settings on page 40.
Text Settings
You can specify the font, size, color, and style for free text, annotations, plots, and
statistics views. In contrast to the Inspector, text settings apply to all objects in the
Experiment document while Inspector text settings apply only to selected objects.
You cannot change text settings of marker, region, or gate labels and definitions.
1 Select the text or views you want to change.
2 Choose Text Settings from the Edit menu (z-T) to access the Text Settings
dialog box (Figure 2-8).
3 Choose a font and point size.
40
BD CellQuest Pro Software User’s Guide
Figure 2-8 Text Settings dialog box
4 Click any combination of Style checkboxes.
Select Condense to compress spacing between characters; select Extend to
stretch out spacing. If both are selected, they cancel each other.
5 Select a font color.
Choose from the 24 available colors. You can change the available color
selections using the Color Palette, as described on page 42.
6 Select the Background and Justification.
•
A Transparent background reveals overlapping items in the text field; an
Opaque background hides overlapping items in the text field. See
Figure 2-5 on page 34.
•
Justification applies to the alignment of free text or annotations within a
text box; it cannot be applied to the text in plots or statistics views.
Select Left, Center, or Right alignment.
Chapter 2: Becoming Familiar with CellQuest Pro Software
41
Color Palette
You can select and apply 24 colors to text, histogram fill, contours, overlaid
histograms, and 3D plots in an Experiment document. Use the Color Palette to
change the available colors.
NOTE Color is not an option if you are using a monochrome or gray-scale
monitor; the palette contains varying shades of gray.
1 Choose Color Palette from the Edit menu.
2 Click on the color you want to change to access the Color Wheel dialog box.
42
BD CellQuest Pro Software User’s Guide
Select colors based on hue, lightness, and saturation (HLS); hue, saturation,
and value (HSV), cyan, magenta, yellow, and black (CMYK); or red, green,
and blue (RGB) color models. Use the HTML Picker to select colors that are
compatible with the Web.
Choose a color on the color wheel for HLS or HSV colors; move the sliders
for RGB, HTML, or CMYK. Each color value ranges from 0% to 100%.
Hue is determined by an angle value instead of % value.
3 Specify the new color and click OK.
The color palette reflects your selection.
4 Click Apply when you are satisfied with the 24 palette colors.
This saves the new colors in the palette, but does not close the window.
5 Click the close box to close the window.
NOTE You must click Apply before closing the window or color changes are
lost. These color changes apply to the current Experiment document; default
settings return when you open another document.
Banners
You can add a standard BD banner or a custom banner of your own design to an
Experiment document. Design a banner in any graphics program and copy and
paste it into CellQuest Pro. Position the banner anywhere on the page and specify
which pages of a multipage document receive the banner. The banner will never
cover an object; objects and windows can overlap the banner, but the banner will
not overlap an object.
1 Create a custom banner in a third-party application and copy it to the
Clipboard.
Use any graphics program that can copy items to the Clipboard as a PICT.
Chapter 2: Becoming Familiar with CellQuest Pro Software
43
2 Choose Banner from the Edit menu.
3 Select Custom to add your own banner, or select Becton Dickinson to insert
a BD banner.
If you are adding a custom banner, paste it into the Picture Preview box
where indicated (Figure 2-9).
Figure 2-9 Banner options
4 Click Positioning to specify the banner position on the page.
•
A dialog box appears with the banner on a scaled page. Drag the banner
to position it.
•
The time and date stamp appear if you checked the checkboxes; drag
these if you want to change their position.
•
Click OK to return to the Banner dialog box.
5 Specify options for multiple pages.
You can limit where the banner appears or have it appear on all pages.
6 Click Apply to see the banner on your document.
44
BD CellQuest Pro Software User’s Guide
7 Close the dialog box when you are satisfied with the appearance of your
document.
NOTE The banner and the time and date stamps can be positioned or
removed only from within the Banner window; they cannot be changed
from within the Experiment document itself. The time and date stamps
update each time the document is opened, save, or printed and when Apply
is clicked.
Grids
If you want to display a grid in the Experiment document, use the Grid command
to set the grid size and determine the units of measure. A grid is saved in the
Experiment document file and restored when the document is opened; it will not
appear on printed Experiment documents.
The unit settings specified in the Grid dialog box are used by the Inspector (see
Changing Object Geometry on page 35).
1 Choose Grid from the Windows menu.
2 Specify grid options in the Grid dialog box.
•
Choose inches or centimeters from the
Grid Size pop-up menu; enter the
grid size within 0.10–2.00 inches or
0.25–5.08 cm.
•
Check Show Grid to display the grid
within the Experiment document view
area. Choosing Snap to Grid from the Windows menu does not display
the grid; you must check Show Grid in the Grid dialog box.
•
Check Snap to Grid to have view area objects snap to the nearest grid
point. See the following section for more information.
Chapter 2: Becoming Familiar with CellQuest Pro Software
45
3 Click OK.
The Experiment document appears with the grid displayed as a pattern of dots
(Figure 2-10).
Figure 2-10 Experiment document view area with grid
Snap to Grid
The Snap to Grid command affects how you create and move objects by restricting
cursor movement to a selected increment (inches or centimeters) within a grid.
This option is helpful if you are trying to align objects precisely.
Snap to Grid does not move objects automatically to align them; you must select
each plot, statistics view, or text object you want to align, and then move or resize it.
You can also use the Inspector to align objects or to make a group of objects the
same size. See Changing Object Geometry on page 35.
1 Choose Snap to Grid from the Windows menu.
A checkmark next to the menu option indicates Snap to Grid is turned on.
Alternatively, choose Grid from the Windows menu, and select the Snap to
Grid checkbox (Figure 2-11).
46
BD CellQuest Pro Software User’s Guide
menu
checkbox
Figure 2-11 Turning on Snap to Grid
2 Select the object you want to move.
When you move an object, the upper left corner snaps to the grid.
NOTE If you add new plots, statistics views, or text annotations, the new
objects will not be snapped to the grid when they first appear; you must
select each object and move or resize it to align it to the grid.
3 Choose Snap to Grid from the Windows menu to turn it off.
Alternately, choose Grid from the Windows menu and uncheck the Snap to
Grid checkbox.
Chapter 2: Becoming Familiar with CellQuest Pro Software
47
48
BD CellQuest Pro Software User’s Guide
3
CellQuest Pro Plots
• becoming familiar with different plot types
• creating plots
• formatting plots
• formatting histograms
• saving Experiment documents
49
CellQuest Pro allows you to acquire or collect data from a flow cytometer, then
analyze the data. During acquisition and analysis, data is displayed in plots. Data
plots can be set up to display all or a selected subset of the data. This section
describes CellQuest Pro plots.
There are three types of plots in CellQuest Pro.
• Acquisition plots can consist of histograms, dot plots, or density plots. These
plots are used to display data while optimizing instrument electronics and to
define the conditions for data collection and storage. To perform acquisition,
you must first create a plot.
• Analysis plots can consist of histograms, dot, density, contour, or 3D plots.
During analysis, a saved data file is displayed in one or more plots. Data can
be analyzed by creating regions, setting markers, and generating statistics.
• Acquisition-to-analysis plots can consist of histograms, dot plots, or density
plots. These plots change from an acquisition plot to an analysis plot after
data acquisition is complete. The data from the file just acquired appear in
the plot with any formatting defined during acqusition, for example,
regions, markers, or colors.
Dot Plot
A dot plot provides a two-parameter data display.
Each dot in a dot plot represents one or more
events (cells or particles). The dot location is
defined by two values, one for each parameter.
Dot plot formatting options are described on
page 56.
Figure 3-1 Dot plot
50
BD CellQuest Pro Software User’s Guide
Contour Plot
Like a dot plot, a contour plot provides a twoparameter data display. Each event has a position
in the plot according to its values for both
parameters. Contour lines in the plot provide a
third dimension by joining x- and y-coordinates
with similar event counts. These plots are similar
to topographic maps which use contour lines to
show points at the same elevation.
Figure 3-2 Contour plot
Contour plot formatting options are described on
page 58.
Density Plot
A density plot simulates a three-dimensional
display of events. It is similar to a dot plot except
it uses colors to show the number of events. As
CellQuest Pro acquires more events, the number
of events change and the colors in the density plot
change. The position of each event on the x- and
y-axis reflects its parameter values. The color
shows the number of events at each x and y
position on the plot.
Figure 3-3 Density plot
Density plot formatting options are described on
page 60.
Chapter 3: CellQuest Pro Plots
51
Histogram
A histogram plot is a graphical means of
presenting a single parameter of data. The
horizontal axis of the graph represents the signal
intensity of the parameter, and the vertical axis
represents the number of events (counts).
Histogram formatting options are described on
page 65.
Figure 3-4 Histogram plot
3D Plot
Like a contour plot, a 3D plot provides a twoparameter display of data. The same data
displayed in a contour plot can be shown from a
different perspective in a 3D plot. Figure 3-5
shows the same data file as that displayed in
Figure 3-2 as a contour plot.
3D plot formatting options are described on
page 62.
Figure 3-5 3D plot
Creating Plots
You can create plots by choosing the desired plot type from the Plots menu or by
drawing a plot using the appropriate tool. You can also duplicate any plot by
choosing the Duplicate option under the Edit menu. Plot options are specified in
the plot Inspector that appears when you create the plot. The contents of the plot
Inspector vary depending on which type of plot is created, but all plot Inspectors
contain Basic Plot, Geometry, Text Style, and FCS Keyword options. Inspector
options for individual plot types are described in Formatting Plots on page 56.
52
BD CellQuest Pro Software User’s Guide
1 Choose the desired plot type from the Plots menu.
The plot and its corresponding Inspector appear in the Experiment
document. Figure 3-6 shows an example of a plot Inspector.
2 Alternatively, draw a plot using one of
Contour
the plot tools.
3D
Using a plot tool allows you to specify
the size and location of the plot.
Density plot
•
Select the appropriate plot tool from the tool palette.
•
Click in an unused section of the Experiment document.
•
Drag diagonally until the plot outline is the desired size.
Histogram
Dot plot
The corresponding plot Inspector appears where you can select plot
options. See Figure 3-6 for an example.
pop-up control
Figure 3-6 Example of a plot Inspector
Chapter 3: CellQuest Pro Plots
53
3 Select the Plot Type in the plot Inspector.
Click once on the Plot Type pop-up control under Basic Plot and choose
Acquisition, Analysis, or Acq –> Analysis from the menu that appears. If you
create an Analysis plot, you can choose Select File from the File pop-up
menu and select a data file to display in the plot.
4 Choose the X and Y parameters.
Click the Parameter pop-up menus in the Inspector to display the available
choices. You can customize parameter labels in the Acquisition Browser (see
Defining Parameter Labels on page 124) or in the Reagent List (see Using
the Reagent List on page 137).
NOTE Histogram plots are single-parameter plots; the Y parameter is not
available.
5 To select a gate, use the Gate pop-up menu.
For information about gates, see Defining Gates on page 157.
6 Display the plot title by clicking the Show Title checkbox.
7 To specify the plot size and location, use the Geometry options.
See Changing Object Geometry on page 35.
8 Specify the Text Style, if desired.
See Changing Text Style and Color on page 32. The text style applies to the
plot title, axes labels, and axes units.
The new plot appears in the Experiment document according to your
specifications.
54
BD CellQuest Pro Software User’s Guide
Creating Additional Plots
Do the following to create additional plots on the Experiment document.
• Choose a plot type from the Plots menu.
• Draw a new plot using one of the plot tools.
• Select an existing plot and choose Duplicate from the Edit menu.
Inspector attributes can be applied to more than one plot at a time. To resize
multiple plots, for example, use the Geometry:Size options in the Inspector.
• Select the plots to be resized.
• Enter the new plot dimensions in the Size text boxes.
To align multiple plots, use the Geometry:Location options in the Inspector.
• Select the plots to be aligned.
• Enter a distance from the top or left margin.
NOTE To avoid overlapping plots, enter either a Top value or a Left value.
Deleting Plots
1 Click on a plot frame to select it.
Selection handles appear on the four corners of the plot. Only a selected plot
can be deleted.
Chapter 3: CellQuest Pro Plots
55
2 Choose Cut or Clear from the Edit menu or press Delete.
The plot and any associated views (statistics, annotations) are removed. You
can undo the deletion of a plot by choosing Undo from the Edit menu.
Formatting Plots
Plot-specific Inspector options for all plots except histograms are described in this
section. Because histograms have multiple formatting options, they are discussed
in a separate section, Formatting Histograms on page 65.
There are slight differences in the format options available for acquisition and
analysis plots. These differences are described under each plot type where
applicable. For all acquisition plots, the File selection button at the top of the
Inspector is grayed.
The appropriate plot Inspector appears when you create a new plot. To format an
existing plot, select the plot, and choose Show Inspector from the Windows menu
(z-I).
Dot Plots
Use the Dot Plot Inspector to format a dot plot.
pop-up control
Figure 3-7 Dot Plot Inspector options
1 Click once on the Show Dots pop-up control and choose an option to
display fewer than all dots.
56
BD CellQuest Pro Software User’s Guide
•
In acquisition, select the number of dots to display (from 100 to All).
For example, if you choose 100, you will see the most recently acquired
100 events.
•
In analysis, select the percentage of dots to display (from 1% to 100%).
When analyzing large data files, you might want to display only a
portion of the events. Displaying 50% of dots for a 2,000-event file will
display every other event and not the first 1,000 events. Statistics are
always calculated on all events in the file regardless of the display.
2 Select the Identify Event checkbox to locate a specific, numbered event on a
plot.
This option is useful when performing single-cell sorting into a 96-well plate
using CloneCyt Plus and a FACSVantage SE flow cytometer. CellQuest Pro
helps you identify a single cell’s position on a plot when the corresponding
clone number is known. For more information, refer to the CloneCyt Plus
User’s Guide.
3 Click the Event Color swatch to specify the dot color.
Select any color in the color selection box that appears. To create custom
colors, see Color Palette on page 42.
4 Select the MultiColor Gating checkbox to display gated data in different
colors.
For information about multicolor gating, see page 160.
Chapter 3: CellQuest Pro Plots
57
Contour Plots
Use the Contour Plot Inspector to format a contour plot.
Figure 3-8 Contour Plot Inspector options
1 Click once on the Scale pop-up control and choose an option to change the
scale.
CellQuest Pro has three methods of calculating contour lines: linear density,
logarithmic density, and probability. The data used by contour plots have a
resolution of 64 x 64 channels. The data from adjacent channels are added to
condense higher resolution data into 64 channels. Statistics calculated on a
contour plot use data in the original resolution, either 256 or 1024.
Choose:
•
Linear Density, and enter a % value, to calculate contour lines as a
percentage of the maximum event number (peak height) and to space
contour lines equally (linearly). The starting value (the outermost
contour) will be half the value you requested.
Example: Enter 20% linear density.
The outermost contour represents 10% of the peak height, the next
contour represents 30%, then 50%, 70%, and 90%. Equal spacing
tends to put most of the contour lines on the higher peaks (representing
larger numbers of events) and might not show lower features.
•
58
Log Density, and enter a % value, to calculate contour lines as a
percentage of the maximum event number and to space contour lines
logarithmically. Log-density contours begin at the percentage of peak
height you entered and continue until they have reached a threshold
value or 1.
BD CellQuest Pro Software User’s Guide
Example: Enter 50% log density.
The innermost contour represents 50% of the peak height, the next
contour represents 50% of the first contour (or 25% of peak height),
and so on. The corresponding percentages based on peak height are
50%, 25%, 12%, 6%, 3%, and 1%. This method shows more detail in
the lower regions, while still showing high peaks.
•
Probability, and enter a % value, to draw contour lines as a percentage of
the total event number. The contour lines are not based on maximum
number of events. Instead, the area between each pair of contour lines
contains an equal percentage of the total events. The starting value (the
outermost contour) is half the value you entered.
Example: Enter 20% probability.
The outermost contour represents 10% of the total number of events,
the next contour represents 30%, then 50%, 70%, and 90%.
2 Select the Smoothing checkbox and enter a value to decrease irregularities in
the contour plot profile.
Enter a value from 1 (minimum smoothing) to 5 (maximum smoothing).
Smoothing does not affect calculated statistics on contour plots.
3 Select the Threshold checkbox and enter a value to set a threshold level for
the outermost (lowest) contour line.
Enter a value from 0% (no threshold) to 100% (maximum threshold). A
threshold provides a level of discrimination to eliminate unwanted data and
is a percentage of the peak. Contour lines below this threshold are not
shown. Thresholding does not affect statistics.
4 Click on the Color swatch to specify the line color.
Select any color in the color selection box that appears. To create custom
colors, see Color Palette on page 42.
Chapter 3: CellQuest Pro Plots
59
5 Select the Multicolor checkbox to display each contour line in a different
color.
The first, innermost contour line is drawn with the second color in the color
palette, the second line with the third color, and so on.
Density Plots
Use the Density Plot Inspector to format a density plot.
Figure 3-9 Density Plot Inspector options
1 Click once on the Scale pop-up control and choose an option to change the
scale.
CellQuest Pro has three methods of calculating density levels: linear density,
logarithmic density, and probability. The data used by density plots have a
resolution of 128 x 128 channels. The data from adjacent channels are added
to condense higher resolution data into 128 channels.
NOTE Probability scaling is not available during acquisition.
Choose:
•
Linear Density, and enter a % value, to calculate density levels as a
percentage of the maximum event number (the peak height) and to
space density levels equally (linearly). The starting value (the lowest
level) will be half the value you requested.
Example: Enter 20% linear density.
60
BD CellQuest Pro Software User’s Guide
The lowest level represents 10% of the peak height, the next level
represents 30%, then 50%, 70%, and 90%. Equal spacing tends to put
most of the density levels on the higher peaks (representing larger
numbers of events) and might not show lower features.
•
Log Density, and enter a % value, to calculate density levels as a
percentage of the maximum event number and to space density levels
logarithmically. Log-density levels begin at the percentage of peak height
you entered and continue until they have reached a threshold value or 1.
Example: Enter 50% log density.
The highest level represents 50% of the peak height, the next level
represents 50% of the first level (or 25% of peak height), and so on. The
corresponding percentages based on peak height are 50%, 25%, 12%,
6%, 3%, and 1%. This method shows more detail in the lower regions,
while still showing high peaks.
•
Probability, and enter a % value, to calculate density levels as a
percentage of the total event number (available only during analysis).
The density levels are not based on the maximum number of events.
Instead, the number of events between each level is the same. The
starting value (the outermost color) is half the value you entered.
Example: Enter 20% probability.
The lowest level represents 10% of the total number of events, the next
level represents 30%, then 50%, 70%, and 90%.
2 In acquisition, enter the maximum event level, or peak, of dots to display
(from 1 to 999,999).
NOTE Peak is not available during analysis.
3 Select the Smoothing checkbox and enter a value to decrease irregularities in
the density plot profile.
Enter a value from 1 (minimum smoothing) to 5 (maximum smoothing).
Smoothing does not affect calculated statistics on density plots.
NOTE Smoothing is not available during acquisition.
Chapter 3: CellQuest Pro Plots
61
4 Select the Threshold checkbox and enter a value to set a threshold level for
the outermost (lowest) density level.
Enter a value from 0% (no threshold) to 100% (maximum threshold). A
threshold provides a level of discrimination to eliminate unwanted data and
is a percentage of the peak. Data below this threshold are not shown.
Thresholding does not affect statistics.
NOTE Thresholding is not available during acquisition.
5 Click on the Color swatch to specify the density level color.
Select any color in the color selection box that appears. To create custom
colors, see Color Palette on page 42.
If a single color is selected (MultiColor left unchecked), all levels are
displayed in different shades of the same color. The color gets lighter at
higher levels.
6 Select the Multicolor checkbox to display the density levels in different
colors.
The first density level is drawn with the second color in the color palette, the
second level with the third color, and so on.
3D Plots
Use the 3D Plot Inspector to format a 3D plot.
Figure 3-10 3D Plot Inspector options
62
BD CellQuest Pro Software User’s Guide
1 Enter new values to change the 3D View Angles.
•
Enter a number between 0 and 90 degrees to change the tilt angle of the
plot.
•
Enter a number between –360 and +360 degrees to change the rotation
angle. The plot rotates around an imaginary axis at right angles to, and
through the center of, the 3D plot grid.
The default rotation is 315; to rotate to the left, enter a number greater
than 315; to rotate to the right, enter a number less than 315.
2 Select the Smoothing checkbox and enter a value to decrease irregularities in
the 3D plot profile.
Enter a value from 1 (minimum smoothing) to 5 (maximum smoothing).
Smoothing does not affect calculated statistics on 3D plots.
3 Select the Threshold checkbox and enter a value to set a threshold level.
Enter a value from 0% (no threshold) to 100% (maximum threshold). A
threshold provides a level of discrimination to eliminate unwanted data and
is a percentage of the peak. Data below this threshold are not shown.
Thresholding does not affect statistics.
4 Click on the Line Color swatch to specify the line color.
Select any color in the color selection box that appears. To create custom
colors, see Color Palette on page 42.
Plot Parameters
You can change X and Y parameters using the Parameter pop-up menus in the
Inspector. Alternatively, you can change parameters directly on any plot except
overlaid histograms and 3D plots.
Chapter 3: CellQuest Pro Plots
63
1 Place the cursor over the plot axis to be changed.
2 Click the mouse button to display a pop-up menu listing the parameters.
The cursor changes to the pop-up cursor whenever it is in the x- or y-axis
area.
pop-up cursor
pop-up menu
Using the Time Parameter
All acquisition plots are time dependent since events are displayed as they come
from the cytometer. For kinetic experiments, you can introduce time as a
parameter in any acquisition plot (dot, density, or histogram).
To enable the time parameter, a time limit must be specified under Collection
Criteria for acquisition and storage settings. See Defining Global Acquisition &
Storage Settings on page 115.
• A dot plot with a time parameter shows events acquired over time. The time
interval you set for acquisition determines the plot axis. The dot plot shows
dots moving left to right, assuming time is the X parameter.
• A density plot with a time parameter is similar to a dot plot with a time
parameter, with the addition of two features:
•
64
The density plot keeps a history of the acquisition. It adds events to the
latest time interval while keeping the information from previous time
intervals.
BD CellQuest Pro Software User’s Guide
•
The plot colors the dots according to the number of events at a
particular channel location.
• A histogram with a time parameter becomes a bar plot showing data
acquired during each time interval. It maintains a history of all events
acquired throughout the acquisition, assuming you have chosen to show all
events. See the following section on formatting histograms.
Formatting Histograms
Use the Histogram Inspector to format a histogram.
Figure 3-11 Histogram Inspector options
1 Select the Log Y Axis option to change from linear to logarithmic scaling.
When the checkbox is unchecked, the y-axis will have a linear scale; when
the checkbox is checked, the y-axis will have a logarithmic scale.
2 Select the Manual Scale checkbox and enter a value to specify the maximum
y-axis value.
Enter a value from 1 to 32,000. If the box is unchecked, the y-axis
automatically scales to the highest peak in the histogram.
3 Click once on the Show Events pop-up control and choose the number of
events to display.
•
In acquisition, select from 100 to All. For example, if you choose 100,
you will see only the last 100 acquired events.
•
In analysis, all events are automatically shown.
Chapter 3: CellQuest Pro Plots
65
4 Select the Smoothing checkbox and enter a value to decrease irregularities in
the histogram profile.
Enter a value from 1 (minimum smoothing) to 5 (maximum smoothing).
NOTE Smoothing is not available during acquisition.
5 Click on the Color swatches to specify outline and fill colors.
Select any color in the color selection box that appears. To create custom
colors, see Color Palette on page 42.
Creating Overlaid Histograms
An overlaid histogram plot is a plot containing more than one histogram. You can
overlay histograms showing any parameter from any file. The initial histogram
appears in the back, with the overlays in front in the order they were created. An
overlaid histogram cannot be saved as an FCS file.
Figure 3-12 Two-dimensional overlaid histogram
1 Select a histogram or overlaid histogram plot.
2 Choose Overlay from the Plots menu to access the Histogram Overlay
dialog box (see Figure 3-13 on page 67).
66
BD CellQuest Pro Software User’s Guide
3 Click Select File to access the Open a Data File dialog box.
Figure 3-13 Histogram Overlay dialog box
4 Locate and select the file to overlay, and then click Open.
The Histogram Overlay dialog box shows the selected file.
5 Specify formatting options for the overlaid histogram; then click OK.
The first four fields in the dialog box—Plot Source, File, Parameter, and
Gate—describe the histogram to be added. The fields in the lower half of the
dialog box apply to all histograms in the overlaid histogram plot with the
exception of overlay color.
After clicking OK, the overlaid histogram appears.
Formatting Overlaid Histograms
After creating an overlaid histogram, you might want to change the formatting.
1 Select the overlaid histogram to reformat.
Chapter 3: CellQuest Pro Plots
67
2 Choose Format Histogram Overlay from the Plots menu (z-F).
selection
arrows
3 Change the order of the overlays, if necessary.
•
Click on the selection arrows to the left of the file name.
•
While holding down the mouse button, drag the file name to the desired
position in the list.
The first file appears in the back, with subsequent files overlaid on top in
the order they are listed.
4 Change the histogram color and line style of any overlay, if desired.
If you are using a black-and-white printer, click the Line Style box so the
overlaid histograms will be printed in different line styles to differentiate
them. A dialog box appears with 10 line style options. Click the desired
style.
5 Select an Overlay View.
68
•
Select Normal to display plots in a two-dimensional view.
•
Select 3D View to display plots in a three-dimensional view.
BD CellQuest Pro Software User’s Guide
•
For 3D View, you can enter values for 3D Tilt and Rotation.
Enter a number between 0 and 90 degrees to change the tilt angle of the
plot.
Enter a number between –360 and +360 degrees to change the rotation
angle. The plot rotates around an imaginary axis at right angles to, and
through the center of, the 3D plot grid.
The default rotation is 315; to rotate to the left, enter a number greater
than 315; to rotate to the right, enter a number less than 315.
Figure 3-14 shows the same histogram in 2D and 3D view.
Figure 3-14 Overlaid histogram, 2D (left) vs 3D (right) view
NOTE When the histogram is in 3D view, markers and regions are not
shown and cannot be added.
6 Select the Log Y Axis option to change the y-axis from a linear to logarithmic
scale.
7 Select a Normalization option.
Normalization applies only to the display of data. It does not affect the
actual number of events in a histogram.
•
Select Global Maximum to normalize the vertical scale to the highest
value found in any histogram in the plot. All histograms appear in their
true relation to each other.
Chapter 3: CellQuest Pro Plots
69
•
Select Each Histogram to normalize each histogram to itself. Peak
heights in each histogram do not necessarily appear in their true relation
to other histograms.
•
Select Manual Scale to assign an absolute value to the vertical scale.
Enter a number from 1 to 32,767 in the designated field. All histograms
appear in their true relation to each other.
8 Click OK to apply your formatting options to the displayed plot.
Displaying Histogram Legends
A legend applies only to overlaid histogram plots; it lists all histogram names,
parameters, gates, and associated colors in the selected plot. Because of space
constraints, individual histograms are not labeled in the plot; the legend allows you
to identify the histograms.
1 Select an overlaid histogram plot.
2 Choose Legend from the Plots menu to access the histogram legend.
legend for histogram in Figure 3-14
You cannot edit this view. To delete the legend, select it and choose Cut or
Clear from the Edit menu, or press Delete.
Using Histogram Tools
Histogram tools can be used to smooth or scale histograms or to add or subtract
channel counts.
1 Select a histogram.
70
BD CellQuest Pro Software User’s Guide
2 Choose Histogram Tools from the Plots menu to access the Histogram Tools
dialog box.
selection
arrows
3 Select the histograms to change by clicking the selection arrows.
To select more than one histogram, click each histogram selection arrow
while holding down the z key (discontinuous selection) or click the first
histogram and last histogram in the list while holding down the Shift key
(continuous selection).
4 Enter a value for Smoothing Iterations to decrease irregularities in the
histogram profile.
•
Enter a value of 1 (minimum smoothing) to 5 (maximum smoothing).
•
Click Smooth.
5 Enter a value for Scale to multiply the channel counts.
•
Enter a value from 0.1 to 99.9.
•
Click Scale.
New histograms replace the old ones. Scaling affects histogram and K-S
statistics.
If you have histograms with different event counts and want to normalize
them, you must reset the scale. Consider the following example:
Chapter 3: CellQuest Pro Plots
71
Histogram A has 10,000 events and histogram B has 8,000 events.
Divide 8,000 by 10,000 to determine a scaling factor of 0.8. Select
histogram A, enter 0.8 in the “Scale by” field, and click Scale. Now both
histograms have 8,000 events and can be added or subtracted.
m CAUTION
Do not use Scale to adjust the y-axis height. Use the histogram
Inspector for a single histogram or choose Format Histogram Overlay from
the Plots menu for an overlaid histogram.
6 Add or subtract channel counts, if desired.
•
Click Add to sum the channel counts. A third histogram is created that
is the sum of the two added histograms. You must name and select a
storage location for the added histogram. The original histograms are
unchanged and the new added histogram appears in the same plot. If
two histograms with different parameters are added or subtracted, the
parameter in the histogram list will be undefined.
•
Click Subtract to subtract the channel counts. If the resulting channel
count is a negative number, it is assigned a channel count of zero. You
must name and select a storage location for the subtracted histogram.
The original histograms are unchanged, and the new subtracted
histogram appears in the same plot. The order in which the two
histograms are selected is not important. The highlighted histogram
nearer the bottom of the list will be subtracted from the one above it.
Reorder the histogram list, if necessary, by clicking the selection arrows
and dragging.
If you do not know the number of events in each histogram, you can display
histogram statistics to obtain the counts.
You can calculate statistics on added and subtracted histograms. See
Chapter 8, Histogram Statistics.
7 Click Delete to remove an overlaid histogram.
The files created by adding or subtracting histograms are not deleted; only
the histogram images are removed from the plot.
72
BD CellQuest Pro Software User’s Guide
Saving Experiment Documents
Choose Save from the File menu to save your Experiment document to a file on
your hard disk. An Experiment document file can contain any combination of
plots, regions, gates, markers, statistics, acquisition tube lists in the Browser, and
calculated expressions. In addition, the Experiment document file retains all
customization options: document size, text and color settings, free text and
annotations, banners, and grids.
When saving an Experiment document, CellQuest Pro saves a reference to the
data files used in the Experiment document, not the data files themselves. See
Saving Data on page 75 for information about preserving data file links. Note that
instrument settings are not saved with the Experiment document; settings are
automatically saved in the linked FCS data file or can be saved separately (see
Saving Instrument Settings on page 100).
The appearance of data in plots or statistics views is not affected when you save an
Experiment document. However, when you open a saved Experiment document
containing acquisition plots, the plots and their associated statistics views are
cleared. Experiment documents containing analysis plots will display data in the
plots unless you specifically instruct CellQuest Pro to ignore the data (for example,
when creating an analysis template).
Saving Experiment Documents as Templates
A CellQuest Pro Experiment document can be saved as a generic template so it can
be reused for routine acquisition or analysis work.
1 Set up a routine acquisition or analysis Experiment document.
The document can have a combination of acquisition and analysis plots with
regions, gates, markers, statistics, colors, and banners defined. You can create
a generic analysis plot by choosing the parameters and gates in the Plot
Inspector without selecting a data file.
Chapter 3: CellQuest Pro Plots
73
2 If any analysis plots have data files associated with them, make the plots
generic by choosing No File from the File pop-up menu in the Inspector.
3 Save the document with a name like “Analysis Template,” then click the
close box.
4 Open the folder where the document was saved and click once on the
document icon (
).
5 Choose Get Info from the File menu (z-I).
6 Select the Stationery Pad checkbox, and close the dialog box.
checkbox
The Experiment document icon changes to show that the document is now
a Stationery Pad:
. You can open the document to acquire or analyze data
while preserving the original template. Each time you open the saved
Experiment document, the statistics views and plots will appear exactly as
you left them.
74
BD CellQuest Pro Software User’s Guide
Using an Experiment Document Template
1 Open the document template.
2 For analysis plots, select the plots, and choose Select File from the File
pop-up menu in the Inspector.
Select the file you want to analyze; adjust markers and regions as necessary.
3 For acquisition plots, choose Connect to Cytometer; then click Acquire.
Saving Data
Data files are automatically saved as separate FCS data files following acquisition
(see Data Files on page 24). Experiment documents displaying data from the file
preserve a link to the file when they are saved. If you move an Experiment
document to a different disk, locate the data files and the Experiment document in
the same folder on the new disk to help preserve data file links.
If a data file used in an Experiment document is moved or deleted and cannot be
found when the document is opened, a message appears. Any plots and statistic
views using that data file appear without data.
• Click OK to dismiss the message box.
• Move the missing data files into the same folder as the Experiment
document; then try opening the Experiment document again.
You can also save a subset of the data in a separate FCS data file. See Saving FCS
Files on page 215.
Chapter 3: CellQuest Pro Plots
75
76
BD CellQuest Pro Software User’s Guide
4
Optimizing Instrument Electronics
• performing instrument quality control
• using CellQuest Pro instrument controls
• optimizing for biological samples
• saving instrument settings
• checking instrument status
• sorting with FACSCalibur, FACSort, or FACSVantage SE
77
Before collecting data, you must optimize instrument electronics for your sample
type. This chapter describes how to use CellQuest Pro instrument controls to
optimize instrument electronics before acquisition.
Before Beginning This Chapter
You should:
• be familiar with general flow cytometer operation. Refer to your instrument
manual for instructions.
• be familiar with Experiment document components and views. Review
Chapter 2.
• know how to create and format plots and how to save Experiment
documents. Review Chapter 3.
Performing Instrument Quality Control
An instrument quality control (QC) procedure, performed on a regular basis,
provides a standard for monitoring instrument performance over time. A general
instrument QC procedure consists of the following:
• running QC particles such as CaliBRITE™ beads to generate instrument
settings
We recommend running FACSComp™ software on a daily basis to perform
photomultiplier tube (PMT) setup, compensation adjustment, and
sensitivity tests. Refer to the FACSComp Software User’s Guide for
instructions. You can use FACSComp calibration files as a starting point to
optimize samples in CellQuest Pro.
• recording settings on an instrument QC log
Sample logs are provided in your instrument user’s guide or training manual,
or you can generate one for your use.
Refer to your cytometer manual or the FACSComp Software User’s Guide for more
information about instrument QC.
78
BD CellQuest Pro Software User’s Guide
Instrument Controls in CellQuest Pro
Before collecting sample data, you must optimize instrument electronics for the
samples being run. During sample optimization, the following instrument controls
are adjusted to optimally display cell populations of interest on the data plots:
• detectors
• amplifiers
• threshold
• compensation
Each instrument control is described in the following sections. To skip to the
optimization exercise, turn to page 85.
Adjusting Instrument Controls
To access instrument controls, you must be connected to the cytometer.
1 Choose Connect to Cytometer from the Acquire menu (z-B).
2 Choose any of the instrument controls to display the associated control
window.
Each window is described in detail in the sections that follow.
•
Choose Detectors/Amps (z-1) to adjust detector
and amplifier settings (described on page 80).
•
Choose Threshold (z-2) to select threshold
parameters and values (described on page 83).
•
Choose Compensation (z-3) to adjust one of
the compensation networks (described on
page 84).
3 Use the pop-up slider controls to adjust the settings within each window.
Chapter 4: Optimizing Instrument Electronics
79
There are three ways to change settings using these controls.
•
Click the up and down arrows. The number is increased or decreased by
one. Click and hold the arrows for faster adjustments.
•
Click the up and down arrows while pressing the Option key. The
number is increased or decreased by ten. Click and hold the arrows for
faster adjustments.
•
Click the icon between the up and the down arrows to display a slider.
Drag the slider up or down. The new value appears in the settings box.
After releasing the mouse button, the slider disappears.
NOTE Detectors/Amps, Threshold, and Compensation windows can vary by
instrument. The examples shown in this chapter are from a FACSCalibur. Refer to
the appropriate instrument manual for other options.
Detectors
Light signals are generated as particles in a fluid stream pass through the laser
beam. These light signals are converted to electronic signals (voltages) and then
assigned a channel number on a data plot. Channel numbers range from 0 through
255 or from 0 through 1023, depending on the chosen resolution. Detectors and
amplifiers adjust these signals so they appear in an appropriate place on data plots.
The two types of signal detectors in the flow cytometer—a photodiode and
photomultiplier tubes—are described in the following sections. Amplifiers are
described on page 82.
Photodiodes
The photodiode is less sensitive to light signals than a PMT, and is therefore more
suitable for detecting the stronger FSC signal. FSC photodiode signals are
amplified using the Voltage pop-up menu in the Detectors/Amps window.
1 Choose Detectors/Amps from the Cytometer menu (z-1) to access the
Detectors/Amps window (Figure 4-1).
80
BD CellQuest Pro Software User’s Guide
This window has three different sizes. To resize the window, click the zoom box.
Figure 4-1 Detectors/Amps windows for the FACSCalibur
2 Click the Voltage pop-up menu and choose a setting.
E00
Multiplies the signal by 100 or 1
E01
Multiplies the signal by 101 or 10
E02
Multiplies the signal by 102 or 100
E03
Multiplies the signal by 103 or 1,000
E-1
Multiplies the signal by 10-1 or 0.1
E01, E02, and E03 increase the signal of small events; E-1 reduces the signal
of large events.
Chapter 4: Optimizing Instrument Electronics
81
Photomultiplier Tubes
Photomultiplier tubes (PMTs) are used to detect the weaker signals generated by
SSC and all standard or optional fluorescence channels. These signals are amplified
by applying a voltage to the PMTs. As the voltage is increased, the detector
sensitivity increases, resulting in increased signal. As the voltage is decreased, the
detector sensitivity decreases, resulting in decreased signal. PMT voltages are
adjusted using the Voltage sliders in the Detectors/Amps window.
1 Choose Detectors/Amps from the Cytometer menu (z-1) to adjust signal
detector voltages.
2 Adjust detector voltages from 150 to 999 V using the Voltage sliders.
See page 79 for instructions on adjusting the slider controls.
Amplifiers
Adjust amplifier settings to make fine adjustments to signals. Choose from linear
or log amplification.
• Log amplification is often used to analyze samples with a large dynamic
range of fluorescence signals. The log scale has four decades of range.
• Linear amplification is usually used for all light scatter parameters. It is also
useful for fluorescent parameters used in DNA quantitation experiments.
Linear amplifier gain can be adjusted from 1.00–9.99. The chosen value
multiplies the signal by a factor of 1.00–9.99.
1 Choose Detectors/Amps (z-1) from the Cytometer menu.
2 Click the Mode pop-up menu and choose Lin or Log.
3 For linear mode, use the Amp Gain sliders to adjust the amplifier gain.
See page 79 for instructions on adjusting the slider controls.
82
BD CellQuest Pro Software User’s Guide
Threshold
Set a threshold to select a channel number below which data will not be processed.
Depending on your instrument and configuration, threshold can be set for one or
two parameters at a time. Only signals with an intensity greater than or equal to
the threshold channel value will be processed and sent to the computer. When
running immunophenotyping samples, for example, the threshold can be set on
FSC to eliminate events such as debris that are smaller than the threshold channel
number. When using propidium iodide (PI) in DNA analysis, threshold can be set
on FL2.
1 Choose Threshold from the Cytometer menu (z-2) to access the Threshold
window (Figure 4-2).
To resize the window, click the zoom box.
Figure 4-2 Threshold windows for the FACSCalibur
2 Click a radio button to select a threshold parameter.
3 Adjust the threshold value using the corresponding slider.
See page 79 for instructions on adjusting the slider controls.
Chapter 4: Optimizing Instrument Electronics
83
Compensation
Compensation allows you to adjust for spectral overlap when samples are stained
with two or more fluorochromes.
Fluorochromes emit light over a range of wavelengths. As a result, a signal from
one fluorochrome may appear in a detector used for another fluorochrome. For
example, FITC appears primarily in the FL1 detector, but some of its fluorescence
overlaps into the FL2 detector. PE appears primarily in the FL2 detector, but some
of its fluorescence overlaps into the FL1 and the FL3 detectors. Compensation
allows you to eliminate this spectral overlap electronically.
1 Choose Compensation from the Cytometer menu to access the
Compensation window (Figure 4-3).
This window has three different sizes. Downsize the window by clicking the
zoom box.
Figure 4-3 Compensation windows for the FACSCalibur
2 Adjust the compensation network using the appropriate slider.
See page 79 for instructions on adjusting the slider controls.
84
BD CellQuest Pro Software User’s Guide
Optimizing the Instrument for Biological Samples
Sample optimization consists of five steps. It is important to perform these steps in
the order in which they are listed.
Procedures for each of these steps are described in the following sections and lysed
whole blood is used as an optimization example. You might have to adapt some of
these procedures for other sample types.
1 Set up a CellQuest Pro Acquisition Experiment document (described on
page 86).
2 Adjust the FSC and SSC detectors and FSC threshold (described on
page 90).
3 Gate on the population of interest; in this case, the lymphocytes (described
on page 91).
4 Adjust the fluorescence detectors (described on page 93).
5 Adjust fluorescence compensation (described on page 96).
When performing sample optimization, prepare proper controls. For example,
include a subclass control to adjust for non-specific antibody binding and
compensation controls to adjust for spectral overlap. In addition, include a normal
sample stained with antibodies to check for normal antibody binding.
Use the subclass control to adjust forward scatter (FSC), side scatter (SSC), and
the FSC threshold; to set a region around the cells of interest; to check FL1, FL2,
FL3, and FL4 settings; and to set quadrants. In our three-color example, the
following subclass control will be used:
Mouse IgG1 FITC/Mouse IgG1 PE/Mouse IgG1 PerCP
Use compensation control samples to adjust the compensation. Each control
should contain a single positive fluorochrome reagent combined with subclass
Chapter 4: Optimizing Instrument Electronics
85
control reagents. Multiple positive fluorochrome reagents can be used if they are
bound to mutually exclusive antibodies. That is, the cells will bind to only one
antibody or the other and none of the cells will bind to both.
In our three-color example, the following compensation controls will be used:
• CD3 FITC/Mouse IgG1 PE/Mouse IgG1 PerCP
• Mouse IgG1 FITC/CD8 PE/Mouse IgG1 PerCP
• Mouse IgG1 FITC/Mouse IgG1 PE/CD45 PerCP
Starting Up the Cytometer
1 Turn on your flow cytometer, and then turn on the FACStation computer.
m CAUTION
To ensure proper communication between the cytometer and the
computer, always turn on the cytometer before turning on the computer.
2 Perform daily instrument quality control (see page 78).
3 Launch CellQuest Pro.
See Launching CellQuest Pro on page 20.
Setting Up an Acquisition Experiment Document
Follow these steps to prepare an acquisition Experiment document.
1 Expand the size of the Experiment view.
•
Choose Document Size from the File menu.
•
Click the square to the right of the highlighted square, and click OK.
See Document Size on page 36 for more details.
86
BD CellQuest Pro Software User’s Guide
2 Create an FSC vs SSC Acquisition dot plot.
See Creating Plots on page 52.
3 Select MultiColor Gating in the Dot Plot
Inspector.
MultiColor gating allows you to view gated events in color.
4 Choose Connect to Cytometer from the Acquire menu (z-B).
The Browser appears. Click on the Acquisition tab, if needed, to show
Acquisition Controls. Click on the title bar and drag the window to a clear
area of the screen.
Figure 4-4 Acquisition controls in the experiment Browser
The Acquisition Browser contains all the tools needed for acquisition. For a
full description, see Using the Acquisition View of the Browser on page 112.
Chapter 4: Optimizing Instrument Electronics
87
5 Choose Detectors/Amps from the Cytometer menu (z-1).
Click and drag the Detectors/Amps window to a clear area of the screen.
6 Choose Threshold from the Cytometer menu (z-2).
.
Click and drag the Threshold window to a clear area of the screen.
Restoring Calib Settings to the Cytometer
To practice sending saved instrument settings to the cytometer, you will retrieve
the Calib file containing instrument settings created by FACSComp and send the
settings to the cytometer. For general information about instrument settings, see
page 99.
88
BD CellQuest Pro Software User’s Guide
1 Choose Instrument Settings from the Cytometer menu to access the
Instrument Settings window.
2 Click Open.
3 Navigate to the Instrument Settings Files folder.
This folder is in the BD Files folder on the FACStation hard drive.
4 Double-click the Calib file.
You can also click once on the file name and then click Open.
The settings change in the Instrument Settings window.
5 Click Set in the Instrument Settings window.
The Calib settings are sent to the cytometer.
NOTE You must click Set to download instrument settings; otherwise,
previous settings will still apply.
6 Click Done.
Chapter 4: Optimizing Instrument Electronics
89
Adjusting FSC, SSC, and FSC Threshold
Adjust the FSC Amp Gain, SSC Voltage, and FSC Threshold to appropriately
display the scatter properties of the sample. For example, Figure 4-5 shows a lysed
whole blood sample.
unadjusted
adjusted
Figure 4-5 Optimizing FSC and SSC
The FSC Amp Gain and SSC Voltage are used to place the population of interest
on scale. FSC Threshold is adjusted to exclude any unwanted debris and noise.
These settings are adjusted in the CellQuest Pro Detectors/Amps window while
running the subclass control.
NOTE You can make these adjustments on any sample tube because the scatter
properties of cells are the same no matter which antibodies are added to the tube.
1 Place the flow cytometer in RUN mode.
2 Place the subclass control tube on the sample injection port (SIP).
For our example, the subclass control is stained with Mouse IgG1 FITC/
Mouse IgG1 PE/Mouse IgG1 PerCP.
3 Click Acquire in the Browser Acquisition Controls (Figure 4-6).
90
BD CellQuest Pro Software User’s Guide
Figure 4-6 Browser Acquisition Controls
Events appear in the FSC vs SSC dot plot. Because Setup is selected in the
Acquisition Control window, you can click Acquire and view a real-time
acquisition display without saving the data to a file.
4 Adjust the FSC Amp Gain in the Detectors/Amps window.
Select a linear gain setting that positions all clusters on scale relative to the
x-axis (see Figure 4-5 on page 90).
5 Adjust the SSC Voltage and/or Amp Gain in the Detectors/Amps window.
For instructions on using the slider controls, see page 79.
Select a linear gain setting that positions all clusters on scale relative to the
y-axis (see Figure 4-5 on page 90).
6 Adjust the FSC threshold in the Threshold window.
Set the threshold to remove most of the debris without cutting off the
lymphocytes (see Figure 4-5 on page 90).
7 Click Pause, then Abort.
8 Remove the subclass control tube from the SIP.
Gating on the Population of Interest
Next, you draw a region around the lymphocytes, the cell population of interest.
Each cell population differs in autofluorescence and nonspecific antibody binding.
Chapter 4: Optimizing Instrument Electronics
91
By gating this population, you can adjust the fluorescence detectors, taking into
account the autofluorescence and nonspecific binding of these cells.
For information about creating and editing regions, see Chapter 6.
1 Select the Polygon-Region tool in the tool palette (
).
The tool becomes highlighted.
2 Draw a polygon region around the lymphocyte population.
•
Move the cursor over the plot; the cursor changes to a crosshair.
•
Position the cursor on the FSC vs SSC dot plot and click to set the first
vertex.
•
Release the mouse button, move the cursor to where you want the
second vertex, and click again.
•
Continue clicking around the population until it is encompassed by the
region.
•
Close the region either by double-clicking or by clicking on the first
vertex.
To edit the region, double-click the polygon outline. The vertices become
handles that can be clicked and dragged to a new location.
Figure 4-7 Region surrounding lymphocytes
92
BD CellQuest Pro Software User’s Guide
Adjusting the Fluorescence Detectors
Mouse anti–keyhole limpet hemacyanin (KLH) antibodies are used in the subclass
control. When these antibodies are added to human cells, any binding that occurs
is nonspecific. Nonspecific antibody binding occurs when the Fc portion of the
antibodies binds to Fc receptors on the cell membrane. This, together with
autofluorescence, is considered background fluorescence.
If necessary, you can reposition the signal from background fluorescence by
adjusting fluorescence voltages on the gated lymphocytes from the previous
section. These adjustments are made in the CellQuest Pro Detectors/Amps
window while running the subclass control.
Create the Plots
1 Create an FL1 vs FL2 Acquisition dot plot.
See Creating Plots on page 52.
2 Choose Gate G1 = R1 from the Gate pop-up
menu in the Inspector.
R1 contains the gated lymphocytes from the
previous section.
3 Click on the frame of the FL1 vs FL2 plot and drag it to the right of the
FSC vs SSC plot.
4 Create an FL3 vs FL2 Acquisition dot plot and choose G1 = R1 for the gate.
Move the plot to the right of the FL1 vs FL2 dot plot.
5 Select the Quadrant-Marker tool in the tool palette (
) to highlight it.
Quadrant markers are set around the gated subclass control data on the
FL1 vs FL2 dot plot. These markers designate areas of negativity and
positivity.
Chapter 4: Optimizing Instrument Electronics
93
6 Click in the FL1 vs FL2 plot and drag the handle of the marker so the
marker is set at 101 in FL1 and 101 in FL2.
Look at the ribbon to see the x- and y-coordinates of the marker. Release the
mouse to set the quadrants. To change the position of the marker, click on
the intersection and drag the marker to a new location. Your plot should
look like the one that follows.
7 Choose Quadrant Stats from the Stats window.
The statistics view appears.
8 To make changes, edit the statistics by choosing Edit Quadrant Stats from
the Stats menu.
94
BD CellQuest Pro Software User’s Guide
Selected statistics will appear in the statistics view. Only the statistics you
select in the Edit Quadrant Statistics dialog box are exported. See Editing
Statistics on page 184 for more information.
Since the statistics view usually contains more information than you need for
spreadsheet analysis, deselect some of the extraneous information. Click in
the box to the left of the statistic to deselect that statistic.
9 Repeat steps 6 through 8 for the FL3 vs FL2 plot.
Adjust the Voltages
1 Install the subclass control tube on the SIP.
2 Click Acquire in the Browser Acquisition Controls.
3 Set the Amp Gain to Log for all fluorescence channels in the Detectors/
Amps window.
4 If necessary, adjust FL1 and FL2 Voltages in the Detectors/Amps window.
Adjust the Voltage until the events are clustered within the lower-left
quadrant of the quadrant markers (Figure 4-8).
unadjusted
adjusted
Figure 4-8 Adjusting the fluorescence detectors
Chapter 4: Optimizing Instrument Electronics
95
5 If necessary, adjust the FL3 voltage to place the negative population in the
lower-left quadrant of the FL3 vs FL2 plot.
Once you have adjusted all fluorescence detectors, events should be aligned
within the lower-left quadrant in the FL1 vs FL3 plot and no further
adjustment should be necessary.
6 Click Pause in the Browser Acquisition Controls.
7 Remove the subclass control tube from the SIP.
8 Click the close boxes of the Detectors/Amps and Threshold windows.
Adjusting Fluorescence Compensation
The last step in the optimization process is adjusting the compensation. Check
and adjust the following compensation networks for 3-color samples, if necessary.
• FL2–%FL1
• FL1–%FL2
• FL3–%FL2
• FL2–%FL3
Compensation controls are used to adjust for spectal overlap and account for
specific binding of antibodies or positive 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. It is important to choose
appropriate reagents for compensation. Base the selection on the panel of reagents
being used. Set compensation using the brightest stained population for each
color. For example, in a panel containing CD3 FITC, CD4 FITC, and CD8
FITC, set FITC compensation using CD8 FITC, the brightest FITC-stained
population in the panel.
96
BD CellQuest Pro Software User’s Guide
Compensation adjustments depend on the detector voltages established for the
subclass control. Once these voltages are set, compensation is adjusted on the
fluorescence-positive cells.
1 Choose Compensation from the Cytometer menu.
Click and drag the Compensation window to a clear area of the screen.
2 Place the CD3 FITC/Mouse IgG1 PE/Mouse IgG1 PerCP tube on the SIP.
3 Click Restart in the Browser Acquisition Controls.
4 If necessary, adjust FL2–%FL1 to place the FITC-positive population in the
lower-right quadrant.
unadjusted
adjusted
5 Remove the tube and place the Mouse IgG1 FITC/CD8 PE/Mouse IgG1
PerCP tube on the SIP.
Chapter 4: Optimizing Instrument Electronics
97
6 Click Pause and then click Restart in the Acquisition Control window.
7 If necessary, adjust FL1–%FL2 to place the PE population in the upper-left
quadrant.
unadjusted
adjusted
8 If necessary, adjust FL3–%FL2 to place the PE population in the upper-left
quadrant.
unadjusted
adjusted
9 Remove the tube and place the Mouse IgG1 FITC/Mouse IgG1 PE/CD45
PerCP tube on the sample handling assembly.
98
BD CellQuest Pro Software User’s Guide
10 Check the FL2–%FL3 compensation.
FL2–%FL3 compensation should not need adjusting because PerCP does
not have much fluorescence in the FL2 channel. Adjust the compensation, if
necessary.
11 Click the close box to close the Compensation window.
12 Click Pause and then click Abort in the Browser Acquisition Controls.
13 Remove the sample tube; then install a tube of DI water on the SIP.
14 Save your instrument settings as described in the following section.
Instrument Settings
Instrument settings are saved automatically in the text section of FCS data files.
However, you can also save settings established for different applications in a
separate instrument settings file. For example, upi cam save the instrument settings
established for instrument quality control (see page 78) to a file and they will be
restored to the flow cytometer the next time instrument QC is performed.
An instrument settings file contains detectors/amps settings, the threshold
parameter and value, compensation settings, pulse processing settings (if applicable
to your cytometer), and special setup values (if applicable to your cytometer).
Chapter 4: Optimizing Instrument Electronics
99
The Instrument Settings dialog box displays a summary of the current settings.
From this window, you can print, save, or restore the settings.
Saving Instrument Settings
1 Choose Instrument Settings from the Cytometer menu to access the dialog box.
2 Click Save.
3 Enter a name (up to 31 characters) in the location dialog box, choose a
location for the settings file, and click Save.
4 Click Done in the Instrument Settings dialog box.
Restoring Instrument Settings
1 Choose Instrument Settings from the Cytometer menu.
2 Click Open.
3 Locate the instrument settings file or FCS file in the location dialog box that
appears.
100
BD CellQuest Pro Software User’s Guide
4 Select the file, and click Open.
The settings change in the Instrument Settings window.
5 Click Set in the Instrument Settings window.
The new settings are sent to the cytometer.
NOTE You must click Set to download instrument settings; otherwise,
previous settings will still apply.
If you want to return to the previous settings, click Revert.
6 Click Done.
Printing Instrument Settings
1 Choose Instrument Settings from the Cytometer menu.
2 Click Print.
The Print dialog box appears.
3 Select the printing options, and click Print.
Checking Cytometer Status
The operating status of the cytometer can be checked anytime during acquisition.
Instrument status can be used as a troubleshooting resource.
There are two ways to check cytometer status: in the BDPAC control panel
window or in the CellQuest Pro Status window.
Chapter 4: Optimizing Instrument Electronics
101
BDPAC Control Panel
The BDPAC is a computer board that allows the computer to communicate with
the cytometer. The BDPAC control panel is used to configure the computer board
for your cytometer type.
During installation, appropriate options are set by your BD service representative.
Change the configuration information only if you connect your computer to
another cytometer, if you reinstall the software, or if you upgrade your cytometer
with new options. You can also access the BDPAC control panel to check
instrument status.
NOTE You must quit CellQuest Pro software to reconfigure the BDPAC control
panel.
1 Choose Control Panels from the Apple menu; then choose BDPAC from the
menu that appears to the right.
NOTE To open BDPAC on G4 computers with Mac OS 9, hold down the
Control and Command keys while choosing the menu item.
2 Verify the instrument status.
The CellQuest Pro Cytometer menu and most Acquire menu items are
disabled if the:
•
computer is turned on before the cytometer
•
communication cable is not connected
•
acquisition card is not present
•
BDPAC is set to None for cytometer type
Some examples of these cytometer problems are shown in Figure 4-9.
102
BD CellQuest Pro Software User’s Guide
Figure 4-9 Examples of cytometer status messages
If the Status indicates a condition you cannot remedy, call the BD Customer
Support Center at (800) 448-2347 in the US. Customers outside the US,
contact your local BD service representative or distributor.
3 Click on the Config button to open the configuration dialog box.
Configuration options vary depending on the cytometer chosen.
FACSCalibur options are shown below; refer to your instrument manual for
other instrument options.
If you are reconfiguring or adding options:
•
Enter the cytometer serial number, if needed.
•
Select the appropriate options.
•
Click OK to close the Configuration window.
Chapter 4: Optimizing Instrument Electronics
103
4 Click the close box to close the BDPAC window; then restart the computer
to make the changes effective.
Status Window
You can use the instrument Status window to check the current state of the
cytometer anytime during a run. You must be connected to the cytometer to
display the Status window. For the FACScan, this is a read-only window. For
FACSCalibur or FACSort, this is also the test pulse controller.
NOTE Instrument status is not available via CellQuest Pro when using a BD LSR
or FACS Vantage family cytometer. Status information appears on the video
monitor of the FACS Vantage or FACSVantage SE; use the BDPAC window to
check the status of the BD LSR cytometer.
1 Choose Status from the Cytometer menu (z-4).
2 Verify the instrument status in the window that
appears.
•
NOT READY could mean that the laser is
warming up, the sheath fluid reservoir is empty, or the waste reservoir is
full. Sheath and waste status are indicated next to Sheath Fluid and
Waste Tank in the status window.
•
STANDBY could mean that the fluidics is set to Standby mode. This is
also referred to as hard standby. If the fluidics is set to RUN mode,
STANDBY could mean that there is no sample on the injection port,
the arm is not under the tube, or the tube is not fully pressurized.
3 Choose FSC, All, or Off from the Test Pulses pop-up menu.
Test pulses are electronically generated signals. On the FACScan and
FACS Vantage family of cytometers, turn test pulses on and off from the
cytometer’s control panel. On the FACSCalibur and FACSort, turn test
pulses on and off in the Status window.
104
•
Choose FSC to generate test pulses in the forward scatter channel only.
•
Choose All to generate test pulses in all channels.
BD CellQuest Pro Software User’s Guide
Sorting
Using the FACSCalibur or the FACSort
Access all sorting controls in CellQuest Pro via the Sort Counters menu item in
the Cytometer menu and the Sort Setup menu item in the Acquire menu. These
menu items are only present if you have a FACSCalibur equipped with the sorting
option, or a FACSort. You adjust instrument settings and set sort gates before
starting the sort. For a complete discussion on sorting, refer to the appropriate
instrument manual.
To set up the sort:
1 Choose Connect to Cytometer from the Acquire menu.
2 Create acquisition plots and define regions for sorting.
You can use any gate for sorting.
3 Choose Sort Setup from the Acquire menu to access the Sort Setup dialog.
4 Choose a Sort Gate from the pop-up menu.
5 Enter the number of cells you want to sort in the Sort Count field.
To sort continuously, enter 0.
Chapter 4: Optimizing Instrument Electronics
105
6 Choose a Sort Mode from the pop-up menu.
Choose among Recovery, Exclusion, and Single Cell. For a complete
discussion on sort modes, refer to the FACSCalibur or the FACSort user’s
guide.
7 Choose whether or not to list aborted events, and click OK.
8 Choose Sort Counters from the Cytometer menu.
In the Sort Counters window that appears, choose the counters to display by
clicking each of the three pop-up menus.
9 Install tubes in the collection station.
To sort:
1 Install a sample on the sample injection port (SIP).
2 Click Acquire in the Acquisition Control window.
Sorting starts immediately. To stop sorting, click Pause, and then click
Abort.
Using the FACSVantage SE
Before acquiring samples or sorting with the FACSVantage SE, you must align and
optimize the cytometer. Before sorting, you must adjust the frequency, amplitude,
and phase of the drop drive, and calculate the drop delay. These adjustments are all
106
BD CellQuest Pro Software User’s Guide
performed directly on the cytometer; refer to the FACSVantage SE instrument
manual. Set the drop delay, deflected drops, sort mode, and direction of unsorted
cells, and define plots and gates. You can make many selections and adjustments
either directly on the cytometer or via the software. However, you must use the
software to set compensation.
To set up CellQuest Pro for sorting:
1 Choose Connect to Cytometer from the Acquire menu.
2 Create acquisition plots and define regions for sorting.
See Chapter 6 for information on defining regions.
3 Choose AutoSORT from the Cytometer menu to access the AutoSORT
window.
4 Follow the steps in the FACSVantage SE manual to enter values for A, B,
and C in this window.
Chapter 4: Optimizing Instrument Electronics
107
5 Click Done when you are finished.
Click Redo if you want to change the current value.
6 Choose Sort Control from the Cytometer menu to access the window.
7 Make selections for Deflected Drops, Sort Modes and Unsorted Cells.
For an explanation of sort modes, refer to the FACSVantage SE manual.
8 Choose Sort Gate Selection from the Acquire menu to access the window.
9 Select left and right sort regions, and click OK.
10 Open the camera door and place collection tubes in the text tube holders.
108
BD CellQuest Pro Software User’s Guide
To sort:
1 Change the Left Sort and Right Sort counters to Count.
2 Press Reset, and close the camera door.
The instrument is now sorting. Continue until the required number of cells
has been sorted. To interrupt the sort, open the camera door.
Chapter 4: Optimizing Instrument Electronics
109
110
BD CellQuest Pro Software User’s Guide
5
Acquiring Data
• using the Acquisition Browser
• defining acquisition conditions
• setting up an acquisition tube list
• using counters
• displaying live statistics
• acquiring data
• using optional acquisition features
111
During acquisition, data is collected from the flow cytometer and displayed in data
plots. Before acquiring data, you must define the events you want to acquire, how
many events to acquire, and where to store the data files. These settings can apply
to the entire Experiment document (global settings) or specified on a tube-by-tube
basis (tube-specific settings).
This chapter describes how to use the Acquisition Browser to define acquisition
and storage settings, how to associate these settings with tubes or experiments, how
to specify acquisition instrument settings, and how to acquire data.
Before Beginning This Chapter
You should:
• be familiar with general flow cytometer operation. Refer to your instrument
manual for instructions.
• be familiar with Experiment document components and views. Review
Chapter 2.
• know how to create and format plots and how to save Experiment
documents. Review Chapter 3.
• have optimized instrument electronics for the sample type to be run. Review
Chapter 4.
Using the Acquisition View of the Browser
The Browser is the command center for monitoring data acquisition and analysis.
There are two views in the Browser: the acquisition view and the analysis view. You
can switch between the two views by clicking on the tabs at the top of the window.
The Analysis view is described on page 196.
• To view the Browser, choose Show Browser from the Windows menu.
• Click on the Acquisition tab, if needed, to show Acquisition Controls.
Alternatively, the acquisition view of the Browser appears when you connect
to the cytometer (z-B) (Figure 5-1).
112
BD CellQuest Pro Software User’s Guide
pop-up control to
add tubes or panels
Figure 5-1 Acquisition Browser
Use the Acquisition view of the Browser to set up and monitor data acquisition.
The Browser lists current parameter settings, all acquisition and acquisition-toanalysis plots in the Experiment document, global and tube-specific acquisition
and storage settings, and a list of tubes to be acquired. You can use the Browser to
set up a list of tubes to be acquired and to monitor the acquisition status. You can
also delete, skip, reorder, or add new tubes to the acquisition tube list.
The following items can be found in the Acquisition Browser:
• Acquisition Controls—a Setup checkbox that, when selected, allows you to
acquire events without saving to a data file; control buttons to start, stop,
pause, or resume acquisition; buttons to specify file name and storage
location; fields where you can enter sample ID, patient ID, and comments;
and fields where you can select or enter Parameter labels. These features are
described in more detail in the sections that follow.
• Global Settings—acquisition and storage settings that apply to all tubes in
an Experiment document except those that have tube-specific settings. Click
once on the disclosing triangle next to the folder to view the icon for global
settings.
Chapter 5: Acquiring Data
113
• Acquisition Plots—icons representing all acquisition and acquisition-toanalysis plots that are not associated with specific tubes. Click once on the
disclosing triangle next to the folder to view the list of plots.
• Acquisition Tube List—list of tubes to be acquired (in order of acquisition),
the file names to be assigned to the tubes, and any plots or settings associated
with the tubes. Click once on the disclosing triangle next to the folder to
view the list of tubes; click once on the triangle next to a tube to view the
settings and plots associated with that tube. Use the pop-up control next to
the Acquisition Tube List folder to add tubes or panels to the list. The
acquisition tube list is described on page 125.
Within the acquisition tube list, the Browser contains:
• Tube-specific settings—has the tube pop-up control to add acquisition and
storage settings or instrument settings specific to that tube. If no settings are
specified, the tube uses global acquisition and storage settings. Tube-specific
settings are described on page 128.
acquisition
pointer
• Acquisition pointer—indicates the tube currently being acquired. You can
drag this pointer to any tube in the list to indicate the next tube to be
acquired.
• Acquisition status icon—indicates the status of each tube in the acquisition
list. The following icons indicate status:
114
◆
Ready for acquisition
✔
Acquisition complete and successful
❊
Acquisition in progress
BD CellQuest Pro Software User’s Guide
Getting Ready to Acquire
Defining Global Acquisition & 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.
Tube-specific settings are described on page 128.
Figure 5-2 illustrates the application of different gates in CellQuest Pro.
• Acquisition gates are specified as part of acquisition and storage settings.
Only events that pass through the acquisition gate are available for data
storage, display in plots, and statistical calculations.
• Plot gates are defined by one or more regions drawn on a plot. A plot gate
defines only the data displayed in the plot and not the data saved to a file.
Regions and gates are explained in Chapter 6.
• Storage gates are also specified in acquisition and storage settings. Events that
pass the acquisition gate and the storage gate are saved in the FCS data file.
The storage gate does not affect the data shown in acquisition plots.
plot gate
acquisition gate
data plot
cytometer
FCS file
events
storage gate
Figure 5-2 Event flow through gates during acquisition
1 Access global acquisition and storage settings by opening the Acquisition &
Storage settings dialog box or Inspector.
•
Choose Global Acquisition & Storage from the Acquire menu to access
the Acquisition & Storage dialog box (Figure 5-3).
Chapter 5: Acquiring Data
115
Figure 5-3 Acquisition & Storage settings dialog box
•
Select the Global Acquisition & Storage Settings icon in the Acquisition
Browser; then choose Show Inspector from the Windows menu. You can
also press (z-I) to access the Inspector (Figure 5-4) or double-click the
Global Acquisition & Storage Settings icon.
Acquisition
Browser
icon
Acq &
Storage
Inspector
Figure 5-4 Acquisition & Storage Inspector
116
BD CellQuest Pro Software User’s Guide
2 Specify the Acquisition Gate.
Inspector
Acquisition & Storage
dialog box
•
Choose whether to accept or reject events that fall within a particular
gate.
•
Specify the events in the gate by choosing from All events or from gated
events (G1 = R1, for example).
3 Specify the Collection Criteria.
Use Collection Criteria to stop collecting events after a certain number of
events have been acquired (event count) or a certain amount of time has
elapsed (time limit).
•
To specify an event count, enter the number of events to collect in the
Events field (Inspector) or Acquisition will stop when field (dialog box).
Make sure the Collection Criteria drop-down menu in the dialog box is
set to Event Count. Select a gate if you want acquisition to stop when
the specified number of events are in the gate.
Inspector
Acquisition & Storage
dialog box
Note that the collection criteria will not change the number of events
stored in the data file. For example, if you want to acquire until you get
5,000 events in G1=R1 and no storage gate is applied, the data file will
contain the 5,000 events as well as the events that were not in the gate.
Chapter 5: Acquiring Data
117
•
To specify a time limit using the Inspector, select the Time Limit
checkbox and enter the desired time. To specify a time limit using the
dialog box, choose Event Count or Time from the Collection Criteria
drop-down menu and enter the desired time in the OR after field.
CellQuest Pro will stop collecting data after acquiring the specified
number of events or after the specified amount of time has elapsed,
whichever comes first.
Inspector
Acquisition & Storage
dialog box
Before every acquisition begins, CellQuest Pro compares the number of
events to acquire with the available storage space to verify there is
enough space for all data. Even when you want to acquire events based
on time, you must enter a number in the event count field so
CellQuest Pro can check for storage space. To make sure the collection
criteria is based on time, enter a number of events greater than you
would expect to collect in this amount of time so the time will expire
before the event count is reached.
•
Choose a Time Resolution value from 10 ms to 10 sec to stop collecting
data after a specified time. Time resolution is the time interval used for
each channel. For example, if the time resolution is 100 ms, you see all
the events acquired during a 100-ms time interval in one channel.
The total acquisition time and the time resolution are related; you can
either enter the total acquisition time and have CellQuest Pro calculate
the time resolution, or you can choose a time resolution and have
CellQuest Pro calculate the total time. If the calculated time resolution
falls between two values shown in the pop-up menu, the greater of the
two values is used.
118
BD CellQuest Pro Software User’s Guide
The following formula shows this relationship:
(time resolution) x (1024 resolution) = total time
NOTE If you pause a timed acquisition, saving of events to the data file is
suspended, but the elapsed time continues.
4 Choose a storage gate, if desired.
Inspector
Acquisition & Storage
dialog box
Specify the events to be stored in the data file—all or gated events. The
storage gate does not limit the data shown in acquisition plots. See
Figure 5-2 on page 115.
5 Select the parameters to save and their resolution.
Inspector
Acquisition & Storage
dialog box
•
Choose a resolution of either 256 or 1024; you cannot have mixedresolution parameters.
NOTE Always choose 1024 resolution if the time parameter will be saved in
data files. Choosing 256 results in a mixed-resolution file that might not be
compatible with other software programs.
•
Specify the parameter information to save in the data file by selecting the
parameters to be saved. You can save disk space by deselecting
unnecessary information.
Chapter 5: Acquiring Data
119
The available parameters depend on the cytometer type and
configuration. See the following section for information on selecting the
parameters to save.
In the Inspector, click the checkboxes to select or deselect parameters. In
the Acquisition & Storage dialog box, click the Parameters Saved
button; then specify parameters in the dialog box that appears. Click
OK to return to the Acquisition & Storage dialog box (Figure 5-5).
Figure 5-5 Selecting parameters to save in Acquisition & Storage dialog box
NOTE To enable the time parameter, a time limit must be specified under
Collection Criteria.
6 Click OK in the Acquisition & Storage dialog box to apply your settings.
Inspector settings are automatically updated.
Selecting Parameters to Save
The parameters used in a plot are defined by number rather than by name. To
work successfully with acquisition-to-analysis plots, it is important to understand
CellQuest Pro’s parameter numbering system.
Acquisition-to-analysis plots can display any parameter analyzed by a cytometer as
well as the time parameter. When saving a subset of a cytometer's parameters to a
data file, the parameter numbering changes. This can affect the parameters
displayed by the plots.
120
BD CellQuest Pro Software User’s Guide
Consider the following example for a FACSCalibur cytometer.
1 Select P1, P3, and P5 in the Parameter Saved dialog box (Figure 5-6).
Figure 5-6 Parameters Saved dialog box
On the cytometer, these parameters are listed as P1, P3, and P5. When they
are saved to the data file, however, they will be listed as P1, P2, and P3 so no
gaps will occur in the numbering. See the following table.
Cytometer (Live Data)
Data File (Saved Data)
Parameter
Number
Parameter
Name
Parameter
Number
Parameter
Name
1
FSC-H
1
FSC-H
2
SSC-H
2
FL1-H
3
FL1-H
3
FL3-H
4
FL2-H
5
FL3-H
6
FL2-A
7
FL2-W
Chapter 5: Acquiring Data
121
2 Create an acquisition-to-analysis dot plot of P1: FSC-H vs P2: SSC-H.
3 Create an acquisition-to-analysis dot plot of P3: FL1-H vs P5: FL3-H.
When the P1 vs P2 dot plot changes to an analysis plot upon completion of
acquisition, it updates using parameters 1 and 2 from the data file.
Parameter 2 for the data file (FL1-H) is not the same as parameter 2 for the
cytometer (SSC-H).
Since there is no fifth parameter in the data file for this example, the P3 vs
P5 dot plot must use parameter numbers that are in range. If one or both
parameters of a dot plot (or density plot) are out of range, the plot changes
its parameter numbers to P1 and P2. The parameter for a histogram plot
changes to P1.
In this example, therefore, the P3 vs P5 dot plot becomes a P1 vs P2 plot and
shows FSC-H vs FL1-H for the data file.
Defining File Name and Storage Location
Use the acquisition view of the Browser to define the file name, storage location,
and parameter labels.
1 Choose Show Browser from the Windows menu.
2 Click on the Acquisition tab, if needed, to display the acquisition view.
3 Click once on the disclosing triangle next to Acquisition Controls, if needed,
to view acquisition controls (Figure 5-7).
122
BD CellQuest Pro Software User’s Guide
Figure 5-7 Acquisition Controls in Browser
4 Click the Change button next to Directory to change the file storage
location.
A location dialog box appears. Navigate to the folder where you want to
store the files; then click the Select button at the bottom of the dialog box.
BD recommends that you do not save files directly to a file server or remote
computer. Move them at a later time.
5 Click the Change button next to File to specify the file name in the File
Name Editor dialog box.
6 Choose a File Name Prefix and File Name Suffix from the pop-up menus.
Chapter 5: Acquiring Data
123
•
Choose Custom Prefix, Sample ID, or Patient ID for the prefix. Enter a
custom prefix of up to 27 characters in the File Name Editor box; enter
a sample ID or patient ID in the Acquisition Browser window.
To use the Load Sample feature, you must enter a unique Sample ID or
Patient ID before acquiring samples
•
Choose File Count, Tube Number, or None for the suffix. If you choose
File Count, CellQuest Pro appends an .nnn to the file name, where nnn
is the number of files acquired since starting acquisition. You can change
the starting number of the File Count by entering a number in the File
Count field. Choose Tube Number if you are going to use a panel for
acquisition (see Creating Panels on page 138). The tube number field is
appended to the file name.
7 Click OK; your choices appear in the Acquisition Browser.
8 Enter comments in the Browser.
Enter up to 32,000 characters. The text automatically scrolls when it reaches
the end of a line. These comments are saved in the data file.
NOTE If you export this field, note that some applications have difficulty
processing text lines longer than 63 characters.
Defining Parameter Labels
Parameter labels can be selected or defined in the Browser.
• Select an existing parameter label by clicking on
the double-headed arrow icon next to each
parameter label and choosing a label from the
pop-up menu that appears.
• Define a new parameter label by entering the
desired label in the appropriate field. The new
labels are saved with the Experiment document.
124
BD CellQuest Pro Software User’s Guide
You can add or delete a reagent label from a parameter label list by editing the
reagent list, as described on page 137.
Setting Up an Acquisition Tube List
Use the acquisition view of the Browser to define a list of tubes to be acquired,
specify the acquisition order, and associate plots with tubes. You can also use the
Browser to define tube-specific settings. All Acquisition Browser options—
acquisition tube list, acquisition order, and tube-specific plots and settings—are
saved when you save the Experiment document.
1 Click the Acquisition Tube List pop-up menu and choose an option from
the list that appears to the right.
click here to
add tubes or
panels
You can select any combination of the following options.
•
Choose Load Tubes from Panel to add a predefined panel of tubes and
plots (see Creating Panels on page 138).
The Acquisition List folder is renamed with the name of the panel and
the tubes in the panel are listed under the folder.
•
Choose New Tube From Panel to add a single tube from a predefined
panel.
•
Choose New Tube to add a single tube; use the Inspector to define the
tube name and parameter settings (see Editing Tubes Using the
Inspector on page 126).
Chapter 5: Acquiring Data
125
The selected tubes or panels of tubes are listed under the Acquisition List
folder. Click the disclosing triangle next to the Acquisition List folder to hide
or show the list of tubes. Any plots not associated with a tube are listed in the
Acquisition Plots folder (Figure 5-8).
Figure 5-8 Acquisition Browser showing plots and tubes
2 Change the order of acquisition by selecting a tube and dragging it up or
down in the list.
The tubes are listed in the order of acquisition. Change the tube order by
clicking on a tube icon and moving it while holding down on the mouse
button.
3 Delete a tube by clicking on the tube icon, then pressing Delete.
The selected tube is removed from the acquisition tube list.
Editing Tubes Using the Inspector
Use the Inspector to change tube names and parameter labels.
1 In the acquisition tube list in the Browser, select the tube to be edited.
Click on the tube icon to select it.
2 Press z-I to display the Tube Inspector if it isn’t showing.
126
BD CellQuest Pro Software User’s Guide
3 Enter or change the tube name.
4 Choose parameters for the tube.
Use the parameter pop-up menus or
enter a name in each field.
Creating Plots and
Associating 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.
1 Create appropriate acquisition plots in the Experiment document.
See Creating Plots on page 52. Each new plot that is created is automatically
listed under the Acquisition Plots folder in the Browser.
2 Select one or more plots to be associated with a particular tube.
You can select the plots in the Experiment document or select one or more
plot icons in the Browser. Hold down the Command key (z) to select
multiple plots that are not listed next to each other in the plot list.
3 Click the Tube pop-up menu in the Inspector and choose a tube.
The Plot Inspector automatically appears after a new plot is created. If you
need to display the Inspector, press z-I.
Chapter 5: Acquiring Data
127
All tubes in the acquisition tube list
are shown.
After choosing a tube, the selected
plots are specific for that tube.
When the tube is acquired, live data
will appear in the designated plots.
4 Repeat steps 2 and 3 for the remaining tubes in the panel.
Defining Tube-Specific Settings
You can define tube-specific acquisition and storage settings and select a different
set of instrument settings for specific tubes in the acquisition tube list. If tubespecific settings are not defined, the tube will be acquired using global acquisition
and storage settings and the current instrument settings for the Experiment
document. All tube-specific settings defined in the Acquisition Browser can be
saved with the Experiment document.
Defining Tube-Specific Acquisition and Storage Settings
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.
pop-up control
Figure 5-9 Adding tube-specific acquisition and storage settings
An Acquisition & Storage Settings icon is added below the tube name. Click
once on the disclosing triangle next to the tube name to display the icon, if
needed.
128
BD CellQuest Pro Software User’s Guide
3 Select the Acquisition & Storage Settings
icon and press z-I to display the
Acquisition & Storage Inspector (or doubleclick the icon).
4 Make any desired 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 Press z-I to hide the Inspector.
Defining Tube-Specific Instrument Settings
Once you have defined instrument settings for your experiment (see Restoring
Instrument Settings on page 100), all tubes in the experiment are acquired using
these settings. However, you also have the option of defining tube-specific
instrument settings. CellQuest Pro creates a reference to one or more alternative
instrument settings files according to your definition. These references are saved
with the Experiment document.
1 Click once on the pop-up control next to a tube in the tube list.
2 Choose Add Instrument Settings from the menu that appears.
pop-up control
Figure 5-10 Adding tube-specific instrument settings
Chapter 5: Acquiring Data
129
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.
3 Select the Instrument Settings icon and press
z-I to display the Instrument Settings
Inspector (or double-click the icon).
4 Click the File button and select the desired 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 Press z-I to hide the Inspector.
Using Counters
CellQuest Pro provides several different types of counters so you can monitor the
progress of acquisition. You can leave the Counters window open during
acquisition to view a running total of the number of acquired events.
1 Choose Counters from the Acquire menu.
The Counters window appears (Figure 5-11). This window has two sizes. To
resize the window, click the zoom box. The additional counters in the
enlarged window apply only if you are acquiring gated data.
130
BD CellQuest Pro Software User’s Guide
Figure 5-11 Counters window, two sizes
2 Choose the Source for each counter.
Click and hold the Source and Mode boxes to open pop-up menus of
available options. You can choose among three different sources for each
counter.
Each Source corresponds to a gate chosen in Acquisition & Storage settings;
the gate label appears between the Source and Mode pop-up menus. If you
change the gates in the Acquisition & Storage settings, the gates in the
Counters window are updated.
•
Choose Accept to count the events that pass through the gate specified
in the Acquisition Gate section of Acquisition & Storage settings. The
Source reads Reject if a reject gate is set. If no gate is defined, this count
is the same as the total event count.
•
Choose Collect to count the events that pass through the gate specified
in the Collection Criteria section of Acquisition & Storage settings.
•
Choose Store to count the events that pass through the gate specified in
the Storage Gate section of Acquisition & Storage settings.
3 Choose the Mode for each counter.
•
Choose Accumulate to display a running total of the events counted.
•
Choose Rate to display the number of events counted over one second.
Chapter 5: Acquiring Data
131
•
Choose % of Total to display the number of events counted as a
percentage of the total number of events coming from the cytometer.
•
Choose % of Accept to display the number of events counted as a
percentage of the number of events that pass through the Acquisition
Gate set in the Acquisition & Storage dialog box.
Displaying Live Statistics
CellQuest Pro can display region, histogram, and quadrant statistics while
acquiring data. First you must create regions or markers; then you can display the
statistics. Live statistics can be displayed as last second or cumulative. See
Chapter 6 for information on creating regions; see Chapter 7 for information on
creating markers and displaying statistics.
1 Select an acquisition or acquisition-to-analysis plot with defined markers or
regions.
2 Choose the statistics to be displayed from the Stats menu.
The corresponding statistics view appears. Live statistics are displayed as data
is acquired.
NOTE Undefined values are represented by *** in a statistics view.
132
BD CellQuest Pro Software User’s Guide
Acquiring Data
Once you have defined acquisition and storage settings and an acquisition tube
list, you are ready to acquire data.
BD recommends you turn off file sharing during acquisition.
NOTE These instructions are for a FACSCalibur. For other flow cytometers, refer
to the appropriate instrument manual.
1 Choose Counters from the Acquire menu; move the window to a clear area
of the screen.
Click the zoom box to display the
enlarged window if you are acquiring
gated data.
zoom
box
2 Choose Connect to Cytometer from the Acquire menu (z-B), if you are not
already connected.
The Acquisition Browser appears
displaying Acquisition Controls.
3 Deselect the Setup checkbox
under Acquisition Controls.
The Directory and File names change from italics to a plain font, indicating
the next acquisition will save events to a file.
4 Place the flow cytometer in RUN mode.
5 Install the first tube on the SIP; immediately place the support arm under
the tube.
Chapter 5: Acquiring Data
133
6 Wait approximately 5 seconds; then click Acquire.
There is a brief period during which the sample tube is pressurized. It is
important to wait for the pressure in the sample tube to stabilize before you
click acquire.
Acquisition to the file begins. The acquisition pointer in the Browser
indicates the tube currently being acquired. If statistics are displayed (see
page 132), live statistics update as data is acquired. Acquired events are
counted in the Counters window. The system beeps at the completion of
acquisition.
7 Remove the tube after the beep.
8 Place the next tube on the SIP.
9 Wait approximately 5 seconds, and click Acquire; remove the tube after the
beep.
If acquiring tube lists, CellQuest Pro automatically updates the parameter
labels for the next tube and increments the file name suffix. If panels are not
being used, only the file count increments.
10 Repeat steps 8 and 9 for the remaining tubes in the panel.
11 Place a tube of DI water on the SIP.
12 Select the Setup checkbox; place the flow cytometer in STANDBY mode.
13 Close the Acquisition Browser and the Counters window when all tubes
have been acquired.
NOTE This does not disconnect you from the cytometer. To do so, choose
Disconnect from Cytometer from the Acquire menu.
134
BD CellQuest Pro Software User’s Guide
Monitoring Acquisition in the Browser
The acquisition pointer in the Browser indicates the tube currently being acquired.
The tube and its parameter labels appear in the Acquisition Controls section of the
Browser (Figure 5-12). You cannot edit the parameter labels at this point because
the panel is already defined.
parameter
labels for
tube being
acquired
acquisition
pointer
Figure 5-12 Tube being acquired in Acquisition Browser
When acquisition of the first tube is complete, the file name, acquisition pointer,
and the parameter labels automatically increment to the next tube. If acquisition
was successful, a green checkmark appears next to the tube.
If you chose Tube Number for the File Name Suffix (see Defining File Name and
Storage Location on page 122), the tube number is appended to the file name.
After acquiring all the tubes in the panel for the first sample, the Tube Number
returns to 001 for the next sample.
Chapter 5: Acquiring Data
135
Pausing Acquisition
Acquisition can occur only in one Experiment document at a time. If you pause
acquisition, you cannot open or create another Experiment document until you
save or abort acquisition. Any additional open documents are hidden; only the
active acquisition document is shown. Folders in the Acquisition Browser are
locked and all Experiment Components are inactive.
1 Click Pause in the Browser Acquisition Controls.
Resume replaces Pause in the Browser window.
2 Select from the following to continue.
•
Resume—to continue collecting events, adding events to those already
acquired.
•
Restart—to begin collecting events, discarding the events already
acquired.
•
Save—to stop collecting events, saving the events already acquired to the
data file.
•
Abort—to stop collecting events, discarding the events already acquired.
Ending Acquisition
There are three ways to end data acquisition:
• Manually—Click Pause; then click Abort or Save.
• By count—Set an event count in the Acquisition & Storage dialog box.
• By time—Set a time limit in the Acquisition & Storage dialog box.
CellQuest Pro stops acquiring data as soon as one of these conditions is met. You
must either quit CellQuest Pro or choose Disconnect from Cytometer from the
Acquire menu to use a different acquisition software. When you disconnect from
the cytometer, all instrument control windows automatically close and the
Cytometer menu is disabled.
136
BD CellQuest Pro Software User’s Guide
NOTE When you open a saved Experiment document, acquisition plots and their
associated statistics views are cleared.
Optional Acquisition Features
This section describes optional CellQuest Pro features that can be used to
customize or simplify acquisition.
Using the Reagent List
The Reagent List is a list of reagent names that can be used as parameter labels or
elements of a panel. Use the Edit Reagent List command to add new reagents or
remove existing reagents from the Reagent List.
1 Choose Edit Reagent List from
the Acquire menu to access the
dialog box.
2 Choose the parameter label list to
edit from the Label pop-up
menu.
The current choices for that
parameter appear in the box.
Many BD reagents are defined in the default reagent list.
3 Click New to add a reagent name or parameter label to the list.
Enter the reagent name in the dialog box that appears; then click OK.
4 To delete a reagent name or parameter label from the list, select the reagent
name; then click Delete.
5 Click OK when the reagent lists are complete.
Chapter 5: Acquiring Data
137
Creating Panels
A panel is a group of reagents commonly used together in the same experiment.
Acquiring and analyzing panels, rather than individual tubes, can save time
because acquisition and analysis can proceed automatically, updating parameter
labels for each subsequent tube.
Choose Edit Panels from the Acquire menu to access the Edit Panels dialog box
(Figure 5-13). The Edit Panels dialog box contains a list of current panels, a list of
the tubes in a selected panel, and a list of the parameter labels for a selected tube.
To view the tubes for a panel, click the icon to the left of the panel name. Click the
icon to the left of a tube to see the parameter labels. The Tube and Labels lists are
empty if no panel is selected or if more than one panel is selected in the Panels list.
Similarly, the Labels list is empty if no tube is selected or more than one tube is
selected in the Tubes list.
Adding a New Panel
1 Choose Edit Panels from the Acquire menu.
The Edit Panels dialog box appears with an IMK-Lymph panel defined in
the default panel list.
Figure 5-13 Edit Panels dialog box
138
BD CellQuest Pro Software User’s Guide
2 Click the Add button.
A new panel is added to the bottom of the list with
the default name new panel.
3 Enter a new panel name (up to 32 characters).
4 Select the new panel and click the Add button above the tube list.
A new tube is added to the
bottom of the tube list.
5 Enter a name for the tube (up
to 32 characters).
6 Select the tube and choose labels for the parameters.
Use the pop-up menus in the Labels list or enter a name in each field.
7 Click Add above the Tubes list to add another tube to the panel.
8 Enter a name for the tube and define parameter labels.
Chapter 5: Acquiring Data
139
9 Repeat steps 7 and 8 for the remaining tubes in the panel.
Editing Panels
You can change a panel or tube name or a parameter label; reorder panels;
duplicate a panel or tube; or delete panels or tubes.
To edit a panel, tube name, or parameter label:
1 Select the panel, tube name, or parameter label to be changed.
2 Enter the new name (up to 32 characters).
Alternately, you can change a parameter label by choosing a new label from
the pop-up menu to the right of the label.
To reorder panels or tubes:
1 Select one or more panels or tubes in the list.
2 Drag them to a new location.
A small arrow in the space to the left of the reordering icons guides you. Sort
panels into alphabetical order by clicking Sort above the Panels list.
To duplicate panels or tubes:
1 Select a panel or tube.
2 Click Duplicate above the Panels list or Tubes list.
NOTE To copy a panel from one computer to another, simply save an
Experiment document with the panel defined. Open the document on the
other computer. To copy all panels to another computer, copy the
CellQuest Pro Prefs file in the System folder into the System folder on the
140
BD CellQuest Pro Software User’s Guide
other computer. Doing this overwrites previous preference and panel
information
To delete panels or tubes:
1 Select the panels or tubes to delete.
2 Click the Delete button above the panel or tube list.
Defining Custom Keywords
You can define custom keywords to be saved in the FCS data file with your
acquired data. Keywords are written to the text section of an FCS file during
acquisition. When the file is opened during analysis, keywords can be displayed in
the Inspector or the statistics view.
Custom keywords each start with an ampersand (&) and a number to identify
them. If a keyword value is not defined, the keyword is not written to the data file.
1 Choose Custom Keywords from the Acquire menu.
2 Click the New button on the Custom Keywords window.
A new line for entering keywords is added. New keywords are automatically
prefixed with an ampersand (&) and a number. There is no limit to the
number of custom keywords, but only the first three can be shown in any
statistics view.
Chapter 5: Acquiring Data
141
If you define two duplicate keywords, the value from the newer keyword is
written to the file and older values from duplicate keywords are deleted.
3 Enter a Keyword of up to 62 characters; press the Tab key to move to the
next field and enter a Value of up to 62 characters.
To delete a keyword, click to the left of the & symbol in the Custom
Keyword dialog box. The entire line is highlighted. Choose Clear from the
Edit menu or press Delete. If you delete keywords from the list, the
remaining keywords are contiguously renumbered.
NOTE If you are acquiring a panel and have created tube-specific keywords,
you will need to redefine the custom keywords before each tube is acquired.
Otherwise, each tube in the panel will contain the same keywords.
Displaying and Exporting Custom Keywords
During analysis, you can display up to three custom keywords in the statistics view
or up to five keywords in any acquisition plot Inspector.
• Select the custom keywords to display in the statistics view using the Edit
Statistics dialog box. See Editing Statistics on page 184.
Any keywords displayed in the statistics view will be exported with the
statistics. See Exporting Statistics on page 216.
• Select up to five keywords (including any custom keywords) to display in the
Inspector by choosing them from the pop-up list in the window. You can
view the value of any FCS keyword in the referenced data file. Click on each
pop-up control to display a list of keywords.
pop-up control
142
BD CellQuest Pro Software User’s Guide
6
Regions and Gates
• creating and editing regions
• using the Region List
• defining a gate
• displaying the Gate List
• using multicolor gates for dot plots
• highlighting dots
143
A region is a boundary you draw around a subpopulation of events on a plot. You
can define up to 16 regions in each Experiment document and use them to define
gates. A gate, which is a combination of regions, is used to isolate subsets of data.
NOTE Regions cannot be drawn on a 3D contour plot or a 3D view of an overlaid
histogram plot.
This chapter provides an overview of the different kinds of regions available in
CellQuest Pro: how to create and edit regions, and how to combine regions into
gates. You should be familiar with regions and gates before analyzing your data.
Before Beginning This Chapter
You should:
• be familiar with Experiment document components and views. Review
Chapter 2.
• know how to create and format plots and how to save Experiment
documents. Review Chapter 3.
Creating Regions
Use the Region tools on the tool palette to create regions on plots.
Histogram Regions
Histogram regions resemble histogram markers on
a histogram plot, except that regions are labeled
with an “R” and markers are labeled with an “M”
(Figure 6-1). Histogram regions are used to define
subpopulations of data for analysis and to define
gates, while histogram markers are used to generate
statistics. For information about histogram
markers, see page 171.
Figure 6-1 Histogram region vs marker
144
BD CellQuest Pro Software User’s Guide
To create a histogram region:
1 Select the Histogram-Region tool in the tool palette (
).
2 Click in the histogram plot to position the left edge of the region; then drag
to position the right edge of the region.
As the cursor passes over the histogram, its shape changes to a crosshair to
indicate that you can draw a region. The ribbon under the title bar displays
the channel number or linear value of the x-axis (X) and the event count (Y)
as you draw the region.
3 Release the mouse button to complete the region.
The region appears with a handle on each endpoint. Click and drag on the
horizontal line of the region to move it; click on either handle and drag the
edge to resize it.
Rectangular, Elliptical, and Polygonal Regions
Draw a region on a dot, density, or contour plot using one of three tools from the
tool palette.
• Rectangular-Region tool—to create a square or rectangular region
• Elliptical-Region tool—to create a circular or elliptical region
• Polygonal-Region tool—to create a free-form region
Figure 6-2 shows these three types of regions.
Chapter 6: Regions and Gates
145
Figure 6-2 Rectangular (R2), elliptical (R5), and polygonal (R6) regions
To create a rectangular or elliptical region:
1 Select the Rectangular- (
) or Elliptical-Region (
) tool in the tool
palette.
2 Click in a dot, density, or contour plot to position the region and drag
diagonally until the region is the desired shape.
As the cursor passes over the plot, its shape changes to a crosshair to indicate
that you can draw a region. The ribbon under the title bar displays the
x and y location of the cursor as you draw the region.
3 Release the mouse button to complete the region.
When you have completed the region, it appears as a solid outline with a
handle on each corner. These handles indicate the region is selected. Click
and drag any of the handles to resize the region. Click on the border of the
region to drag it to a new position.
To create a polygonal region:
1 Select the Polygonal-Region tool in the tool palette (
).
2 Click in a dot, density, or contour plot to position the first vertex of the
polygon region.
146
BD CellQuest Pro Software User’s Guide
3 Move the cursor to the next desired position and click to add another vertex.
A solid line appears connecting the two vertices.
To restart or cancel the region after setting the first vertex, click outside the
plot view.
4 Continue moving and clicking around the population until you reach the
first vertex.
5 Click on the first vertex to complete the polygon.
You can also double-click the mouse button to set the last vertex and
complete the polygon.
When you have completed the region, it appears as a solid outline with
handles at each corner of the polygon. These handles indicate the region is
selected (Figure 6-3). Click and drag any of the handles to resize the region.
Click on the border of the region to drag it to a new position.
Figure 6-3 Polygon region (selected)
NOTE A polygon region can only be concave in one direction (X or Y)
(Figure 6-4). CellQuest Pro prevents you from adding a vertex that would
create an illegal shape or rotating the polygon into an illegal shape.
Chapter 6: Regions and Gates
147
polygon concave in y direction
polygon concave in x direction
Figure 6-4 Concave polygon in x and y directions
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 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.
NOTE Snap-To regions can be created only in Analysis plots.
To create a Snap-To region:
1 Select the Snap-To–Region tool in the tool palette (
).
2 Click on a cluster in a dot, density, or contour plot.
The Snap-To region is automatically drawn
on the plot. Note that the region outline
appears thicker than that of other regions to
differentiate the Snap-To feature.
The region appears with handles at each
corner. These handles indicate the region is
selected.
148
BD CellQuest Pro Software User’s Guide
Snap-To
region
A Snap-To region can be edited just like a polygonal region: click and drag
any of the handles to resize the region; click on the border of the region to
drag it to a new position. See Editing Regions on page 150.
NOTE If the cluster is too small or diffuse for the Snap-To 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.
Reading New Data into a Snap-To Region
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.
When updating, the Snap-To region searches for a cluster in the same area it was
originally placed. If the new cluster is too small, diffuse, or too far away, the SnapTo region remains in its original location.
Copying Snap-To Regions
You can copy a Snap-To region just like any other region in CellQuest Pro (see
Copying Regions on page 155). However, any copies of the region appear and
behave like a standard polygon region; that is, they do not change when a new data
file is read into the plot containing the copied region.
If you edit or move the original Snap-To region you will edit or move all copies of
the region. Similarly, if you edit or move any of the copies, you will edit or move
the original region. Thus, reading a new data file into the plot displaying the
original Snap-To region will affect all copies of the region in the Experiment
document.
Chapter 6: Regions and Gates
149
Editing Regions
You can move or edit the shape of any region. You can edit a polygon region, but
you can cannot add a vertex to an existing region. Region statistics are
automatically updated after a region is edited (see Displaying Statistics on
page 182 for instructions on displaying region statistics). Changes to a region will
apply to all copies of the region in the Experiment document.
1 Click once on any region to select it.
Handles appear at each corner of the region or each side of a histogram
region to indicate it is selected (Figure 6-5).
R3 = Rectangular region
R7 = Polygon region
R6 = Elliptical region
R2 = Snap-To region
R1 = Histogram region
Figure 6-5 Examples of selected regions
2 To move a region, click on the border of a selected region and drag the
border.
Click on the horizontal line of a histogram region to move it.
3 To resize a region, click on one of the handles of a selected region and drag
the handle.
4 To reposition the vertex of a polygon or Snap-To region, double-click on the
region’s border.
150
BD CellQuest Pro Software User’s Guide
Alternatively, click once on the border; then choose Edit Polygon from the
Gates menu.
The selection handles are now on the vertices of the polygon, not the corners
of the rectangle circumscribing the polygon (Figure 6-6).
deselected polygons
selected polygons
edit-mode polygons
Figure 6-6 Deselected, selected, and edit-mode polygons
5 Click and drag a vertex to the desired position.
To exit the edit mode, click outside the region to deselect it or choose Stop
Editing Polygon from the Gates menu.
6 To move a region label, deselect the region; then click and drag the label to
its new location.
Rotating Regions
All regions except histogram regions can be rotated. Region statistics are
automatically updated after rotating a region.
1 Select the region.
2 Choose Rotate Region from the Gates menu.
3 Click and drag one of the handles.
Chapter 6: Regions and Gates
151
The region rotates around the center of the region, not the handles. To exit
the rotate mode, click outside the region to deselect it or choose Stop
Rotating Region from the Gates menu.
Deleting or Hiding Regions
You can delete a region from an Experiment document or hide a region from view.
• Hidden regions no longer appear in the view but still appear in the Region
List (see Using the Region List on page 154) and are still valid in gate
definitions (see Defining Gates on page 157). Hidden regions are still
defined and can be displayed again.
• Deleted regions are removed from the Region List and from any plots where
they appear. Any gate using the region becomes invalid and any plot using
the invalid gate goes blank. In statistics views, data from an invalid gate is
either undefined (denoted as ***) or 0, depending on the statistic. For
example, the total events can be 0, but the CV cannot be 0 and is therefore
undefined. Deleted regions cannot be displayed again.
To delete a region:
1 Click on one or more regions to select them.
To select multiple regions in a plot, click each region while holding down the
Shift key.
2 Press the Delete key or choose Clear from the Edit menu.
The region is removed from the Region List and from all plots in which it
appears.
NOTE You can also delete regions by selecting them in the Region List and
pressing Delete.
152
BD CellQuest Pro Software User’s Guide
To hide a region:
1 Press the Option key while clicking inside a plot.
A pop-up menu appears listing all the regions in
the Experiment document. Regions outside the
selected plot are grayed out (R4, in this
example); regions currently displayed in the plot
are enabled and checked.
2 Choose regions to hide from the pop-up menu.
•
To hide a single region, choose the region
name to uncheck it.
•
To hide all regions, choose Hide All.
3 Alternatively, click on one or more regions in the plot to select them.
4 Press Option-Delete simultaneously.
The regions are hidden on the plot but still exist in the Region List.
To display hidden regions:
1 Press the Option key while clicking inside a plot.
A pop-up menu appears; hidden regions are
enabled and unchecked.
2 Choose a region name from the pop-up menu to
recheck it.
The region reappears on the plot.
3 To show all regions, choose Show All from the
pop-up menu.
Chapter 6: Regions and Gates
153
Using the Region List
Use the Region List to display a list of all regions in an Experiment document and
to define labels for the regions.
Regions are named with a region number in the order they are created (R1
through R16). When you delete a region, the next region you define is given the
lowest R number available. For example, if you have R1, R2, and R3 defined and
you delete R2, the next region you define will be named R2.
1 Choose Region List from the Gates menu.
The Region List window appears showing the name of the associated
Experiment document (Figure 6-7). Click on the zoom box to expand the
window.
selection
arrows
zoom box
Figure 6-7 Region List window
2 To name a region, double-click in the Label field.
3 Enter the new label, using up to 40 characters.
4 Click anywhere outside the Label field to complete the entry, or press Tab or
Return.
154
BD CellQuest Pro Software User’s Guide
The new name appears in the Region List and on all plots using that region.
NOTE If the region label consists of two or more words with spaces between
the words, quotation marks are automatically put around the words in the
Gate List. This allows the software to determine where the region label starts
and ends. If you want to define a gate in the Gate List using a region labeled
with more than one word, you must put quotation marks around the label.
See Defining Gates on page 157.
5 To reorder the Region List, click the selection arrow of the region you want
to move and drag it up or down to a new position on the list.
Copying Regions
You can select one or more regions to copy and paste back into that plot or into
another plot with the same parameters. There are two ways to copy a region: copy
the definition of the region from the Region List, or copy the image of the region
from a plot. There are also two ways to paste a region: paste the copy into a plot, or
paste the copy into the Region List.
Keeping in mind that an Experiment document has only one Region List, you can
copy a region definition and paste it into:
• another plot with the same parameters in the same Experiment document.
The region is drawn in the plot. The region is already defined in the Region
List, so there are no changes to the Region List.
Chapter 6: Regions and Gates
155
• a plot in a different Experiment document. The region is drawn in the plot
and the definition is added to the Region List if there is room.
• a Region List in a different Experiment document. It is added to the list if
there is room, but no images are drawn.
In addition, you can copy a region image and paste it into:
• another plot with the same parameters in the same Experiment document.
The region is drawn in the plot. The region is already defined in the Region
List, so there are no changes to the Region List.
•
a plot in a different Experiment document. The region is drawn in the plot
and the definition is added to the Region List if a region does not already
exist with the same name and if the limit of 16 regions has not been
exceeded. None of the regions in the Clipboard will be pasted if there is not
enough room in the Region List for all of them.
• a Region List in a different Experiment document. It is added to the list if
there is room, but no images are drawn.
In summary, if you are copying a region from one Experiment document to
another and are pasting into a plot, the image is drawn and the definition added. If
you are pasting into the Region List, the definition is added but the image is not
drawn.
See page 149 for information on copying Snap-To regions.
To copy and paste a region:
1 Select either the region in a plot or the region in a Region List.
To select more than one region in a plot, click each region while holding
down the Shift key. To select more than one region in the Region List, click
each region selection arrow while holding down the Command key (z)
(discontinuous selection) or click the first region and last region in the list
while holding down the Shift key (continuous selection).
2 Choose Copy from the Edit menu.
156
BD CellQuest Pro Software User’s Guide
3 Activate the destination plot or Region List, and paste the region in the
desired location.
Keep in mind the following:
•
A histogram region cannot be pasted into a two-parameter plot and a
region from a two-parameter plot cannot be pasted into a histogram.
•
A region cannot be pasted into a plot in a different Experiment
document if the region has the same name as a region in the other
document.
•
You cannot have more than 16 regions in each Experiment document.
Defining Gates
A gate consists of one or more combined regions. Use gates to isolate subsets of
data for analysis or sorting. By default, CellQuest Pro assigns gate 1 to region 1
(G1 = R1), gate 2 to region 2 (G2 = R2), etc. You can modify these assignments
according to your experiment.
Using Combined Regions in Gate Definitions
To define a gate using combined regions, use the logical operators AND, OR, and
NOT. You can use the words or their equivalent symbols in your gate definitions.
R1 AND R2 When the AND (*) operator is used, only events that are in both
(R1 * R2)
regions satisfy the gate.
NOTE 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.
Chapter 6: Regions and Gates
157
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.
You must insert spaces between the logical operators and the definitions. For
example, CellQuest Pro cannot recognize R1ANDR2, but can recognize R1 AND
R2. If the region label consists of more than one word, put quotation marks
around the label in the Gate List.
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. Consider the following
examples:
• NOT R1 AND R2—CellQuest Pro first reads NOT R1, and then
AND R2: all events outside R1 and also in R2.
NOT (R1 AND R2)—CellQuest Pro first reads R1 AND R2, and then
applies NOT: all events outside the intersection of R1 and R2.
• R1 OR R2 AND R3—CellQuest Pro first reads R2 AND R3, and 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 these events and R4.
158
BD CellQuest Pro Software User’s Guide
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.
Using the Gate List
Use the Gate List to label, define, and display up to 16 gates for an associated
Experiment document. By default, CellQuest Pro assigns gate 1 to region 1 (G1 =
R1), gate 2 to region 2 (G2 = R2), etc. If you rename a region, the Gate List is
updated.
1 Choose Gate List (z-G) from the Gates menu.
The Gate List window appears showing the name of the associated
Experiment document (Figure 6-8). Click on the zoom box to expand the
window.
zoom box
selection
arrows
valid gates
invalid gates
(note italics)
Figure 6-8 Gate List window
2 Enter the gate Definition.
Use region labels, not region numbers, in gate definitions. If the region label
contains more than one word, put quotation marks around the label. See
Using the Region List on page 154.
Chapter 6: Regions and Gates
159
For a gate to be valid, the regions that comprise it must be valid, or defined.
All labels and definitions of invalid gates are italicized.
3 To name a gate, double-click in the Label field.
4 Enter the new label, using up to 40 characters.
5 Click anywhere outside the Label field to complete the entry, or press Tab or
Return.
The new name appears in the Gate List.
6 To reorder the Gate List, click the selection arrow of the gate you want to
move and drag it up or down to a new position on the list.
Copying a Gate
Gates cannot be copied. However, the text of a gate label or definition can be cut
or copied and pasted into another Gate List. The regions that comprise the gate are
not copied when you copy the text of a gate label or definition.
Using Multicolor Gates for Dot Plots
For dot plots, you can use the multicolor option to color events that satisfy gates in
the Gate List without actually gating the data. This allows you to visually compare
subpopulations that appear in different dot plots.
When you select multicolor gating for a dot plot, the events take on the colors of
the gates listed in the Gate List according to the following conditions:
• For each event, CellQuest Pro scans down the Gate List, starting with the
first gate listed at the top. An event is drawn in the color of the first valid gate
that is satisfied and that has its Multicolor checkbox checked.
• If a given event does not satisfy any of the gates in the Gate List, it will take
on the color of the gate for the dot plot.
160
BD CellQuest Pro Software User’s Guide
• An event is drawn in the color selected for Single Color if it does not satisfy
any other gate in the list, and there is no gate for the dot plot.
Figure 6-9 illustrates multicolor gating using two dot plot views of the same data
and the associated Gate List, using data file NORM003 from the CellQuest Pro
Sample Files folder.
On the two plots, lymphocytes, monocytes, and granulocytes are colored
according to which gates they satisfy. Notice that the population in G4 is not
colored because the multicolor checkbox is unchecked in the Gate List. Any
populations in undefined or invalid gates (listed in italics) are not colored even if
their multicolor checkboxes are checked.
NOTE You can reorder the gates in the Gate List to change the gate hierarchy, or
priority (and therefore the color) of the events in multicolor dot plots.
Figure 6-9 Multicolor gating example
Chapter 6: Regions and Gates
161
Setting Up a Gate List for Multicolor Gating
1 Choose Gate List from the Gates menu.
2 Click the checkboxes, if unchecked, to select gates for multicolor.
3 Click the color box to change the color of the gate.
The Color Selection dialog box appears with the 24 color choices. Click the
color to make your selection. The new color appears in the color box.
Highlighting Dots
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. Use
the Hilite feature to locate a rare population on a dot plot or to differentiate
between a mixed population within a region.
NOTE Highlighting does not add events nor does it affect statistics.
Figure 6-10 and Figure 6-11 show how checking the Hilite checkbox changes the
selected dot population.
Figure 6-10 Area with mixed population (R3 + R4 events)
162
BD CellQuest Pro Software User’s Guide
Hilite box
checked
Figure 6-11 Area with R3 events highlighted
Chapter 6: Regions and Gates
163
164
BD CellQuest Pro Software User’s Guide
7
Statistics
• calculating data
• creating and editing histogram markers
• displaying histogram statistics
• generating overlaid histogram statistics
• creating and editing quadrant markers
• displaying quadrant statistics
• displaying region and gate statistics
• editing statistics
• using the Expression Editor
165
CellQuest Pro generates statistics from acquired events. The following types of
statistical analysis can be displayed:
• Histogram statistics for an entire histogram or sections of a histogram.
• Kolmogorov-Smirnov (K-S) statistics—a two-sample test to determine if two
histograms come from different populations.
• Region statistics for two-parameter plots; statistics are displayed for all
regions in the Region List.
• Gate statistics for two-parameter plots; statistics are calculated for all valid
gates in the Gate List.
• Quadrant statistics for two parameter plots; statistics are calculated for each
quadrant on the plot.
This chapter contains an overview of the statistical analysis features in
CellQuest Pro; you should be familiar with statistics before analyzing your data.
Before Beginning This Chapter
You should know how to:
• create and format plots and how to save Experiment documents. Review
Chapter 3.
• create regions and define gates. Review Chapter 6.
Calculating Data
For all types of statistical analysis, you can set up calculation to occur
automatically or on demand. You can specify log data units for calculating and
reporting statistics on logarithmically amplified data. CellQuest Pro software can
calculate the percent gated vs. percent total events in a plot and generate means
and geometric means. These general statistical features are described in this
section.
166
BD CellQuest Pro Software User’s Guide
Automatic vs Manual Calculation
When you start CellQuest Pro for the first time, all statistical calculations are
automatic. This means that immediately after you make changes in an Experiment
document, CellQuest Pro calculates any items affected. You can set automatic
calculation to occur only when you want by turning off the Auto Recalculate
feature. This allows you to delay certain time-consuming updates until you are
ready.
Turning Auto Recalculate off suspends some operations that trigger data
processing. These operations include:
• changing the data displayed in a plot
• changing plot parameters
• changing the color of a gate
• adding, deleting, or altering regions and gates
Calculations are not affected by moving, resizing, or zooming plots.
NOTE Some operations cause a partial update of information. For example, if you
change the data file of a dot plot while Auto Recalculate is turned off, the plot axes
and title update, but the data in the plot does not.
1 To turn off automatic recalculation, choose Auto Recalculate from the Gates
menu.
The checkmark in front of the menu item disappears.
automatic
recalculation
on vs off
2 To make a calculation while in the manual mode (Auto Recalculate turned
off ), choose Recalculate Now from the Gates menu.
Chapter 7: Statistics
167
Alternatively, press z= or select the Calculator tool in the tool palette (
).
In manual mode, all calculations are kept in a processing queue until
Recalculate Now is chosen or Auto Recalculate is turned back on.
NOTE Because some information in the Experiment document might not be valid
when items are awaiting calculation, printing, Export Stats, and Append Stats are
not enabled. After recalculating the data, these items are once again enabled.
Selecting Log Data Units
CellQuest Pro uses two methods to calculate and report statistics on logarithmically
amplified data. The first method uses channel values; the second method uses linear
values.
• Channel values correspond to the data resolution value (0–255 or 0–1023).
• Linear values refer to a dynamic range of signal intensities (100–104 or
1–10,000).
The method you select applies to all types of statistics in the entire Experiment
document, not just a particular plot.
All calculations begin with a channel value (or number) because this is how the
cytometer sends digital signals to the computer. If you select Channel Values as the
method of calculation, calculations are performed on the channel numbers and the
results are expressed as channel values.
You might prefer to compare the relative intensities of logarithmically amplified
signals by converting the channel value to a number that corresponds to the
fluorescence intensity. This converted number is referred to as a linear value. If you
select Linear Values as the method of calculation, the channel numbers are
converted to linear values using the following equation:
Linear value = 10(channel number/scaling factor*)
After the conversion, statistics are calculated and the results are expressed as linear
values. Remember that only parameters acquired with log amplification are
*
168
For 256-channel resolution data, the scaling factor is 256/4 or 64. For 1024-channel resolution
data, the scaling factor is 1024/4 or 256, where 4 is the number of log decades.
BD CellQuest Pro Software User’s Guide
affected. CellQuest Pro uses information in the FCS file to identify these
parameters. Parameters acquired with linear amplification are not affected.
To change Log Data Units:
1 Choose Log Data Units from the Plots menu.
The Log Data Units dialog box appears.
2 Click the radio button to change the
current selection; then click OK.
All plots and statistics views are updated
with the new values.
Calculating Percent Gated and Percent Total Events
Percent of gated and percent of total events can be calculated for all types of
CellQuest Pro statistics. These values can be displayed in all statistics views.
• The percent gated is calculated by taking the number of events within a
quadrant, region, gate, or marker and dividing by the number of events in
the gate that was applied to the data.
• The percent total is calculated by taking the number of events within a
quadrant, region, gate, or marker and dividing by the total number of
ungated events in the data file.
For example, assume you have an FCS data file that contains 10,000 events. You
can create a histogram that contains 8,000 events by gating the data. If you create
a marker that encloses 2,000 events, you get the following percentage statistics for
the marker:
percent gated = ( 2000 ⁄ 8000 ) × 100 = 25%
percent total = ( 2000 ⁄ 10,000 ) × 100 = 20%
Chapter 7: Statistics
169
Calculating Means
CellQuest Pro calculates means and geometric means. Geometric means are
preferable when dealing with skewed populations.
The formulas for mean and geometric mean are:
∑X
mean = ------------i
n
∑
geometric mean = 10
log ( X i )
--------------------------n
where Xi is the channel value or the linear value for the ith event and n is the
number of events used in the calculation.
Remember that for data acquired with log amplification, you can convert the
channel values to linear fluorescence values. Statistics can be calculated using linear
or channel values. The formulas shown above are used in either case. Note that the
mean channel value, when converted to a linear fluorescence value using the
formula on page 169, is not the mean intensity value. In fact, averaging the
channels and converting them to linear is the equivalent of calculating the
geometric mean. Be sure to convert to linear fluorescence before calculation if you
want the mean or geometric mean of the linear fluorescence.
The geometric mean can be calculated for any parameter regardless of the data
mode, log or linear, and the log data units settings.
170
BD CellQuest Pro Software User’s Guide
Using Histogram Markers
CellQuest Pro calculates statistics for an entire histogram or for sections of the
histogram. Each section includes a range of channels on the histogram x-axis. The
sections are defined by setting histogram markers.
Histogram markers consist of an upper and lower boundary connected by a
horizontal line. A marker generates statistics for the events in a designated section
of the histogram. You can draw markers only on a single histogram plot or a 2D
view of an overlaid histogram plot. You can define up to eight markers for each
plot. Histogram regions resemble histogram markers on a histogram plot except
that regions are labeled with an “R” and markers are labeled with an “M” (see
Figure 6-1 on page 144).
Creating Histogram Markers
1 Select the Histogram Marker tool in the tool palette (
).
2 Click in the histogram plot to position the left edge of the marker; then drag
to position the right edge of the marker.
As the cursor passes over the histogram, its shape changes to a crosshair to
indicate that you can define a histogram marker. The ribbon under the title
bar displays the channel number or linear value of the x-axis (X) and the
count (Y). For overlaid histograms, the Y value is blank.
3 Release the mouse button to complete the marker.
The marker appears with a handle at each endpoint. Click and drag on the
horizontal line of the marker to move it; click on either handle and drag the
edge to resize it. The marker is added to the Marker List.
Chapter 7: Statistics
171
Editing Histogram Markers
Histogram statistics are automatically updated after editing a histogram marker.
The Marker List is updated with the new location.
1 Click on the horizontal line of the marker to select it.
Handles appear at each
corner of the region or each
side of a histogram region to
indicate it is selected.
2 To move the marker, drag
the horizontal line to a new
location.
selected (M1) vs
deselected (M2)
histogram
markers
3 To resize the marker, click on one of the handles of a selected region and
drag the handle to the desired position.
4 To move a marker label, deselect the marker; then click and drag the label to
its new location.
5 To delete a marker, select it; then choose Cut or Clear from the Edit menu or
press Delete.
If you delete a marker from the Marker List, it is automatically deleted from
the plot. If you delete a marker from the plot, it is automatically deleted
from the Marker List.
Displaying a Marker List
Use the Marker List to display a list of markers in a histogram. You can copy, paste,
delete, reorder, or give a new label to a marker on the Marker List. Since each
172
BD CellQuest Pro Software User’s Guide
histogram plot can have its own markers, you can open a Marker List for each
histogram plot in an Experiment document.
1 Select a histogram plot.
The Marker List applies to that plot only.
2 Choose Marker List from the Gates menu.
The Marker List window appears showing the name of the associated
histogram, a list of all markers in the histogram, and the left and right
boundries of each histogram marker.
selection arrows
3 To name a marker, double-click in the Label field.
4 Enter the new label, using up to 40 characters.
5 Click anywhere outside the Label field to complete the entry, or press Tab or
Return.
The new name appears in the marker list and on the histogram plot using
that marker.
6 To reorder the marker list, click the selection arrow of the marker you want
to move and drag it up or down to a new position on the list.
Chapter 7: Statistics
173
Copying Markers
You can select one or more markers to copy and paste to another plot. There are
two ways to copy a marker: copy the image of the marker from a plot, or copy the
definition of the marker from the Marker List. There are also two ways to paste a
marker: paste into a plot, or paste into the Marker List. If you paste an image, the
definition is automatically added; if you paste a definition, the image is
automatically drawn.
1 Select the marker to be copied from the histogram plot or in the Marker
List.
2 Choose Copy from the Edit menu or press z-C.
The marker is copied to the Clipboard.
3 Click the plot or the Marker List where you want to paste the marker.
4 Choose Paste from the Edit menu or press z-V.
The marker appears in the plot and is added to the Marker List if a marker
with the same name does not already exist and there are less than eight
markers already defined. If you copied more than one marker to the
Clipboard, none of the markers in the Clipboard will be pasted if there is not
enough room in the Marker List for all of them. The two plots do not need
to have the same axis parameters.
Converting Markers to Regions
Markers can be converted to regions that can be used to define gates. Remember,
markers provide statistics for histograms; regions are components of gates used to
define subpopulations of events.
1 Select the marker to be converted in the histogram plot or in the Marker
List.
174
BD CellQuest Pro Software User’s Guide
2 Choose Copy from the Edit menu (z-C).
3 Choose Region List from the Gates menu.
4 Choose Paste from the Edit menu (z-V).
The information is added to the Region List if a region with the same name
does not already exist and there are fewer than 16 regions already defined. Its
image, however, is not drawn on any plots. If you copied more than one
marker to the Clipboard, none of the markers in the Clipboard will be
pasted if there is not enough room in the Region List for all of them.
If you copy marker M1 to the Region List, its label is still M1, not R1. The
default in the Gate List is G1=R1. Therefore, to use M1 in gates, either
change the label in the Region List to R1, or change the definition in the
Gate List to M1.
Displaying Histogram Statistics
Statistical information from a histogram plot is displayed in the Histogram
Statistics window. To display this window:
1 Select a histogram plot.
2 Choose Histogram Stats from the Stats menu.
The Histogram Statistics view appears (Figure 7-1).
Chapter 7: Statistics
175
Figure 7-1 Histogram Statistics view
Undefined values are represented by ***. To display more or less information
in the window, edit the histogram statistics as described on page 184. For a
brief description of the items displayed in the window, see Table 7-1 on
page 185 and Table 7-5 on page 189.
Generating Overlaid Histogram Statistics
There are two statistical options that apply only to overlaid histograms. One
option allows you to select different histograms for the numerator and
denominator and calculate percent gated and percent total events. The other, the
Kolmogorov-Smirnov statistics option, determines if two histograms come from
different populations.
Calculating Percentages From Overlaid Histograms
You can express the number of events within one histogram’s marker as a
percentage of events in a second histogram. In other words, you can calculate
percent gated and percent total events using the number of gated and total events
from a different histogram as the denominators in the equations.
This feature is commonly used with subtracted histograms. The overlaid histogram
plot in Figure 7-2 shows a control histogram (NORM001) that is subtracted from
another histogram (NORM002) to create a third histogram (NORM002–
176
BD CellQuest Pro Software User’s Guide
NORM001). (Instructions for creating subtracted histograms can be found in
Using Histogram Tools on page 70.)
NORM001
NORM002–NORM001
NORM002
Figure 7-2 Subtracted histogram example
Histograms must be overlaid in the same plot to be subtracted. To better illustrate
the subtraction, Figure 7-3 shows the two component histograms and the resulting
subtracted histogram in separate plots.
(minus)
(equals)
Figure 7-3 Test histogram (NORM002) – control histogram (NORM001) = subtracted
histogram
To express the NORM002–NORM001 histogram as a percentage of the
NORM002 histogram, do the following:
1 Select the subtracted plot and choose Histogram Stats from the Stats menu
to access the dialog box (Figure 7-4).
Chapter 7: Statistics
177
Figure 7-4 Histogram Statistics dialog box
2 Select the numerator (NORM002–NORM001) and denominator
(NORM002) histograms; then click OK.
The Histogram Statistics view appears. In this example, there are 27.75%
total events and 67.64% gated events in the subtracted histogram in
comparison to the test histogram (NORM002).
178
BD CellQuest Pro Software User’s Guide
Remember, the selection of numerators and denominators affects only the
percent gated and percent total in overlaid histograms; other statistics are not
affected.
Calculating Kolmogorov-Smirnov (K-S) Statistics
The Kolmogorov-Smirnov (K-S) two-sample test determines if two overlaid
histograms come from different populations. The calculation computes the
summation of the curves and finds the greatest difference between the summation
curves. You can calculate K-S statistics for the whole overlaid histogram or for a
section of the histogram by setting a marker. Both histograms must have the same
data mode, logarithmic or linear. Smoothing and scaling affect K-S statistics.
1 Select an overlaid histogram plot.
2 Choose K-S Stats from the Stats menu.
The Kolmogorov-Smirnov Statistics dialog box appears.
3 Select the two histograms to compare by clicking their names while holding
down the Command key (z).
You can also select a marker to restrict the range over which the statistics are
calculated.
Chapter 7: Statistics
179
4 Click OK.
The Kolmogorov-Smirnov Statistics view appears.
The following statistics are reported:
Channels
The portion of the histogram used in the analysis
D/s(n)
An index of similarity for the two curves. If D/s(n) = 0, the curves
are identical. In this equation, s(n) equals the square root of
(n1 + n2)/(n1 * n2), where n1 is the number of events in the first
histogram and n2 is the number of events in the second histogram.
D
The Kolmogorov-Smirnov statistic; simply, the greatest difference
between the two curves
Channel
Channel number of D (where the greatest difference between the
two curves occurs)
P Value
The probability of D being as large as it is given that the two
selected histograms are from the same population
For a complete discussion of these statistics, refer to the article written by IT
Young in the Journal of Histochemistry and Cytochemistry. 1977;25:935-941.
180
BD CellQuest Pro Software User’s Guide
Creating Quadrant Markers
A quadrant marker divides a 2D plot into four sections (quadrants) for calculating
statistics. A plot can have only one quadrant marker. If you change the parameters
of the plot, the quadrant marker and statistics are removed.
1 Select the Quadrant Marker tool in the tool palette (
).
2 Click in the plot and drag to position the intersection of the quadrant
markers.
As the cursor passes over the plot, its
shape changes to a crosshair to
indicate that you can define
quadrant markers.
The ribbon under the title bar
displays the x and y location of the
cursor as you draw the markers.
ribbon
handle
3 Release the mouse button to complete the marker.
Quadrant markers appear with a handle at the intersection. To move the
intersection, click on the handle and drag it to a new location.
Editing Quadrant Markers
Quadrant statistics are automatically updated after editing quadrant markers.
1 Click on the intersection of the quadrant markers to select them.
A handle appears to indicate the markers are selected.
Chapter 7: Statistics
181
•
To delete the markers, choose Cut or Clear from the Edit menu, or press
Delete.
If quadrant statistics are displayed, they are also deleted.
•
To move the markers, drag the handle to a new location.
•
To copy the markers, choose Copy from the Edit menu or press z-C.
Click the plot where you want to paste the marker; choose Paste from
the Edit menu or press z-V.
The new marker appears. The two plots need not have the same axis
parameters. If a quadrant marker already exists in the plot, pasting
another quadrant marker updates the existing marker to the new
location and updates the associated quadrant statistics.
2 Click anywhere outside the marker intersection to deselect the quadrant
markers.
Displaying Statistics
To generate statistics for a dot plot, contour plot, or density plot, you must first
define quadrants, regions, or gates.
Display statistics by selecting a plot, then choosing the desired
option from the Stats menu.
• Quadrant Stats displays calculated statistics for each
quadrant on the selected plot.
• Region Stats displays calculated statistics for all regions in
the Region List. You can generate region statistics for a
plot even if the regions were drawn on another plot with
different parameters.
• Gate Stats displays calculated statistics for all valid gates in the Gate List.
Figure 7-5 shows an example of each statistics view. Undefined values are
represented by ***. To display more or less information in the window, edit the
histogram statistics as described on page 184. For a brief description of items
displayed in the windows, see Table 7-1 on page 185 through Table 7-4 on
page 188.
182
BD CellQuest Pro Software User’s Guide
Figure 7-5 Examples of statistics views
To maintain statistical integrity, the following rules apply:
• Statistical results cannot be modified.
• You can cut or copy an entire statistics view, but not the individual items in
the view. Individual items can only be extracted for use in calculated
expressions. See Using the Expression Editor on page 191.
Chapter 7: Statistics
183
Editing Statistics
You can specify what information appears in each statistics view by editing the
statistics. You can specify the formatting of the text and location of the view using
the Inspector.
To specify the information appearing in a statistics view:
1 Select the statistics view.
2 Choose Edit (xxx) Stats from the Stats menu.
The menu item changes depending on what type of statistics view is selected.
If you selected the histogram statistics view, for example, the Stats menu
item reads Edit Histogram Stats.
After making your selection, the appropriate Edit Statistics dialog box
appears.
Figure 7-6 Edit Histogram Statistics dialog box
184
BD CellQuest Pro Software User’s Guide
3 Click to place a checkmark in the checkboxes of items you want to appear;
click to remove the checkmark in the checkboxes of items you don’t want to
appear.
A brief description of the items common to all statistics can be found in
Table 7-1. Statistic-specific information is described in Table 7-2 on
page 187 through Table 7-4 on page 188.
4 Click OK in the dialog box.
The statistics view does not automatically resize itself when statistics are
deselected. You can resize it by dragging the selection handles. The new size
is remembered when you get another data file or save the Experiment
document.
Table 7-1 Items available in all statistics views
Header Info
Title Line
Labels the view with the window title (Histogram Statistics,
for example)
File Name
Name of data file
Log Data Units
Units used for calculating and displaying statistics (linear
or channel values)
Sample ID
Sample identification
Patient ID
Patient identification
Patient Name
Patient name
Case Number
Case number
Chapter 7: Statistics
185
Table 7-1 Items available in all statistics views (continued)
Header Info
Custom Keywords
Up to three custom keywords can be displayed in each
statistics view. The default is to display none. A custom
keyword with its value is displayed if you have defined it.
For example, if you select Custom Keyword #1 in the Edit
Histogram Statistics dialog box, but you have not defined
that keyword, then it will be displayed as Custom
Keyword #1: with no value following
Tube
Tube name
Panel
Panel name
Acquisition Date
Date file was acquired or created
Gate
Gate label or No Gate
Total Events
Total number of events in the data file
Gated Events
Number of events in the gate applied to the data file
Parameter
Histogram x-axis parameter and its data mode (linear or
log)
Smoothing Iterations
Always reads zero because statistics are calculated on
unsmoothed data
Quad Location
Labels the view with the x and y location of the quadrant
(Quadrant Stats, only) markers in linear or channel values
Live Stats Events (not available for gate statistics)
Last second or
Cumulative
Calculate live stats for the last second or for all data
accumulated since live acquisition began. This option
disabled when selected view is in Analysis mode. Default
setting is Last second.
Header Columns
1 or 2 Columns
186
Select 1 or 2 header columns for an individual view;
default setting is 2 Columns
BD CellQuest Pro Software User’s Guide
Table 7-2 Statistics specific to Quadrant Statistics
Column Title
(view)
Item (Edit Stats
dialog box)
Quad
Quadrant Label
Inserts a quadrant label before each quadrant
statistic: UL = upper-left; UR = upper-right;
LL = lower-left; LR = lower-right
Events
Event Count
Number of events in each quadrant
% Gated
Percent of Gated
Percentage of events in a quadrant compared
to total events in the gate
% Total
Percent of Total
Percentage of events in a quadrant compared
to all events in the original file
X Mean
X Mean
Average x-axis channel number or linear
value for events in the quadrant
X Geo Mean
X Geometric
Mean
Average of the logarithm of the x-axis
channel number or linear value for events in
the quadrant, expressed as the anti-log
Y Mean
Y Mean
Average y-axis channel number or linear
value for events in the quadrant
Y Geo Mean
Y Geometric
Mean
Average of the logarithm of the y-axis
channel number or linear value for events in
the quadrant, expressed as the anti-log
Description
Table 7-3 Statistics specific to Region Statistics
Column Title
(view)
Item (Edit Stats
dialog box)
Description
Region
Region Label
Region label from region list
Events
Event Count
Number of events in each region
% Gated
Percent of Gated
Percentage of events in the region compared
to number of events in the gate
Chapter 7: Statistics
187
Table 7-3 Statistics specific to Region Statistics (continued)
Column Title
(view)
Item (Edit Stats
dialog box)
% Total
Percent of Total
Percentage of events in the region compared
to total events in the data file
X Mean
X Mean
Average x-axis channel number or linear
value for events in the region
X Geo Mean
X Geometric
Mean
Average of the logarithm of the x-axis
channel number or linear value for events in
the region, expressed as the anti-log
Y Mean
Y Mean
Average y-axis channel number or linear
value for events in the region
Y Geo Mean
Y Geometric
Mean
Average of the logarithm of the y-axis
channel number or linear value for events in
the region, expressed as the anti-log
Px, Py
Region
Parameters
Parameters used in the region (parameter
number of the X and Y axes for each region)
Description
Table 7-4 Statistics specific to Gate Statistics
188
Column Title
(view)
Item (Edit Stats
dialog box)
Description
Gate
Gate Label
Gate label from Gate List
Events
Event Count
Number of events in each gate
% Gated
Percent of Gated
Percentage of events in the gate compared to
total events in the gate applied to the plot
% Total
Percent of Total
Percentage of events in the gate compared to
all events in the data file
BD CellQuest Pro Software User’s Guide
Table 7-5 Statistics specific to Histogram Statistics
Column Title
(view)
Item (Edit Stats
dialog box)
Description
Marker
Marker Label
Marker label from marker list
Left, Right
Marker Bounds
Locations of the left and right boundaries
Events
Event Count
Number of events in each marker of the
selected histogram
% Gated
Percent of Gated
Percentage of events in a marker compared
to total number of events within the gate
% Total
Percent of Total
Percentage of events in a marker compared
to total events in the data file
Mean
Mean
Average channel number or linear value of
events within a marker
Geo Mean
Geometric Mean
Average of the logarithm of the channel
numbers or linear values of the events within
a marker, expressed as the anti-log
SD
SD
Standard deviation: a measure of the spread
about the mean for events within a marker
CV
CV
Coefficient of variation: the SD divided by
the mean within a marker, expressed as a
percentage
Median
Median
Channel number or linear value that divides
a histogram in the marker into two parts
with equal numbers of events
Peak
Peak
Number of events at the highest point
within the marker
PeakCh
Peak Channel
Channel number or linear value of the peak
within a marker
Chapter 7: Statistics
189
Formatting Statistics Views
Use the Inspector to change the font style or the location and size of a statistics
view.
1 Select the statistics view to reformat.
2 Choose Show Inspector from the Windows menu or press z-I.
The Inspector window appears.
3 Make the desired selections in the Inspector window.
Text Style options are described on page 32; Geometry options are described
on page 35.
190
BD CellQuest Pro Software User’s Guide
Using the Expression Editor
Use the Expression Editor to combine statistics in user-defined mathematical
expressions for data analysis. For example, you might want to use quadrant
statistics from a lymphocyte fluorescence plot to calculate T-helper/suppressor
ratios. Expressions update automatically as data in the document changes.
1 Display statistics for the appropriate plot.
2 Select the Expression Editor tool in the tool palette (
).
Alternatively, choose New Expression from the Stats menu; the Expression
Editor appears.
expression box
3 Enter a Label for your custom expression, if desired.
The label will precede your expression in the Experiment document.
4 Click to place the cursor inside the expression box.
Chapter 7: Statistics
191
5 Enter operands for your custom expression.
Operands can consist of the following:
•
Individual statistics from a statistics view—click on the statistic to add it
to the expression box. Expression results will be updated each time the
individual statistic changes.
•
A user-defined constant—use the keyboard to enter.
•
Results of another calculated expression—click on the expression to add
it to the expression box. Expression results will be updated if the other
calculated expression changes.
6 Use the function keys to build your custom expression.
For functions containing parentheses, click inside the parentheses to enter an
operand. Use function keys for the following.
Multiplies the displayed number by the next entered quantity
Divides the displayed number by the next entered quantity
Adds the next entered quantity to the displayed number
Subtracts the next entered quantity from the displayed number
Calculates the natural logarithm (base e) of the number in the
display
Calculates the natural antilogarithm of the number in the display
Calculates the common logarithm (base 10) of the number in the
display
192
BD CellQuest Pro Software User’s Guide
Calculates the common antilogarithm of the number in the display
Isolates numerical expressions for separate calculation; defines
sequence of operation
The result of your custom expression is automatically displayed in the Result
window; invalid or incomplete entries are displayed as ######.
7 Enter a suffix and any comments.
The suffix will appear after your expression in the Experiment document;
comments will appear only in the Expression Editor window. Use the
comments field to keep notes on how the expression was created.
8 Click OK to transfer your custom expression to the Experiment document.
The result of your expression appears in the view area with its label and any
specified suffix. Handles appear at each corner of the expression box to
indicate it is selected (Figure 7-7).
expression in view area
Figure 7-7 Sample expression and appearance in view area
Chapter 7: Statistics
193
Editing Expressions
1 Select an expression in the Experiment document view area.
Handles appear at each corner of the expression to indicate it is selected.
•
Move the expression by clicking on it and dragging it to a new location.
•
Delete the expression by choosing Cut or Clear from the Edit menu or
by pressing Delete.
2 Choose Edit Expression from the Stats menu to make changes to the
expression.
The Expression Editor appears displaying the current expression. Click in
any field to enter new information.
194
•
Click OK to apply your changes and transfer the edited expression to
the view area.
•
Click Clear to erase the current expression.
•
Click Cancel to leave the expression as is.
BD CellQuest Pro Software User’s Guide
8
Analyzing Data
• using the Analysis Browser
• analyzing data
• analyzing groups of data files
• exporting statistics
• using quantitative calibration
195
During analysis, acquired data from FCS data files is displayed in appropriate
plots. You can use regions and gates to restrict your analysis to the populations of
interest. CellQuest Pro analyzes the data displays and calculates statistics that you
can print or export.
This chapter describes how to perform data analysis, analyze groups of data files,
and export statistics. At the end of the chapter, you will find a brief description of
quantitative calibration.
Before Beginning This Chapter
You should:
• be familiar with Experiment document components and views. Review
Chapter 2.
• know how to create and format plots and how to save Experiment
documents. Review Chapter 3.
• be familiar with data acquisition. Review Chapters 4 and 5.
• know how to create regions, define gates, and display statistics. Review
Chapters 6 and 7.
Using the Analysis Browser
The Browser is the command center for monitoring data acquisition and analysis.
You can switch between the two views in the Browser—the Acquisition view and
the Analysis view—by clicking on the tabs at the top of the window. The
Acquisition view is described on page 112.
• To view the Browser, choose Show Browser from the Windows menu.
• Click on the Analysis tab, if needed, to access the Analysis view (Figure 8-1).
196
BD CellQuest Pro Software User’s Guide
Load Sample
pop-up shortcut
Figure 8-1 Sample Analysis Browser
The Analysis Browser provides a hierarchical listing of all panels, tubes, and plots
in the Experiment document, grouped by data file. All plots using the same data
file (or tube) are listed under the name of that tube; all tubes acquired as part of a
panel are listed under the name of that panel. You can use the Browser to navigate
to specific plots in the document, a handy feature when you are using a large view
area with many plots.
The following objects can appear in the Analysis Browser:
• Panels—all tubes referenced in the Experiment document that were acquired
as part of a panel are listed under the name of that panel. Click once on the
disclosing triangle next to the panel name to see a list of tubes in the panel.
Panels are described on page 138.
The Load Sample pop-up control next to the panel name allows you to load
new data files into plots currently displayed under the panel. For a full
description, see Using the Load Sample Feature on page 208.
Chapter 8: Analyzing Data
197
• Tubes—tubes that were acquired as part of a panel are listed under the panel
name. When not associated with a panel, tubes are listed individually with
the name of the tube data file shown in the Filename column. Note the
difference in icons between tubes ( )and panels ( ). Click once on the
disclosing triangle next to the tube name to see a list of plots associated with
that tube.
• Plots—all Analysis plots and Acq–>Analysis plots associated with a tube or
panel are listed under the name of their associated tube. Any plots not
associated with a tube or panel are listed in the uppermost level of the
Browser.
Navigating to Specific Plots
To find a plot in the Experiment document view area, hold down the Option key
while clicking on the plot title in the Browser.
The selected plot appears in the view area.
Analyzing Data
CellQuest Pro comes with several sample data files that you can use to practice
data analysis. Sample files are located in the CellQuest Pro folder inside the
BD Applications folder (Figure 1-1 on page 19). In this section, we will show you
an example of data analysis using the sample files. Alternatively, use these steps as a
guideline to analyze your own data files.
Data analysis consists of the following:
• creating an Analysis Experiment document
• displaying statistics
• using the Next Data File command to analyze other data files
You can practice many of the steps in this section by performing the CellQuest Pro
Analysis Tutorial located on your FACStation CD-ROM.
198
BD CellQuest Pro Software User’s Guide
Using Sample Files for Practice
The Sample Files folder contains 12 data files from three different samples. Each
sample was stained with the four-tube reagent panel listed in Table 8-1.
Table 8-1 Data files in Sample Files folder
sample 1
sample 2
sample 3
Tube 1: γ1 FITC/γ2a PE
NORM001
NORM005
NORM009
Tube 2: CD3 FITC/CD19 PE
NORM002
NORM006
NORM010
Tube 3: CD3 FITC/CD4 PE
NORM003
NORM007
NORM011
Tube 4: CD3 FITC/CD8 PE
NORM004
NORM008
NORM012
This section describes data analysis of a single panel. Once you are familiar with
data analysis, you can set up CellQuest Pro to automatically analyze multiple tubes
in a panel and multiple panels. See Analyzing Groups of Data Files on page 208.
Setting Up an Analysis Experiment Document
1 Launch CellQuest Pro, if you have not already done so.
The software starts up with a new, untitled Experiment document.
2 Expand the Experiment document window to full size by clicking the zoom
box in the upper-right corner of the view area.
3 Choose Show Browser from the Windows menu.
4 Click on the Analysis tab to view the Analysis Browser.
The Experiment Components section is empty; as you create plots in the
Experiment document, they will be added to the Browser.
Chapter 8: Analyzing Data
199
Creating Analysis Plots
1 Create an Analysis dot plot.
See Creating Plots on page 52 for assistance.
2 In the Dot Plot Inspector, select NORM001 for the File; change the Event
Color to a color other than white.
NORM001 is an isotype control data file in the Sample Files folder.
3 Draw a region around the lymphocytes on the NORM001 dot plot.
Use the Snap-To tool to draw a quick and simple region. See Snap-To
Regions on page 148 for assistance. Your dot plot and Inspector should look
like those shown in Figure 8-2. Note that the plot and tube have been added
to the Acquisition Browser.
Figure 8-2 Analysis dot plot with Snap-To region around the lymphocytes
4 Create an Analysis contour plot of FL1 vs FL2 displaying NORM001 gated
data.
5 Make sure the scale is set to Log Density; change the Contour Color to a
color other than white.
200
BD CellQuest Pro Software User’s Guide
6 Draw quadrant markers around the negative population in the contour plot.
Set the marker so that the subclass population is in the lower-left quadrant.
See Creating Quadrant Markers on page 181 for assistance. Your contour
plot and Inspector should look like those shown in Figure 8-3. Note that the
plot has been added to the Acquisition Browser.
Figure 8-3 Analysis contour plot with quadrant markers around negative
population
7 Create an Analysis FL1 histogram displaying NORM001 gated data.
8 Change the Line and Fill colors in the Inspector.
9 Draw a histogram marker around the negative population.
See Creating Histogram Markers on page 171 for assistance.
10 Draw a second histogram marker from the upper boundary of marker 1 to
the end of the histogram plot.
Your histogram, Inspector, and Browser should look like those shown in
Figure 8-4.
Chapter 8: Analyzing Data
201
Figure 8-4 Analysis histogram with markers around negative population,
Histogram Inspector, and Browser
Displaying Statistics
1 Select the contour plot; choose Quadrant Stats from the Stats menu.
The Quadrant Statistics view appears displaying data from data file
NORM001.
2 Select the Quadrant Statistics view and choose Edit Quadrant Stats from the
Stats menu.
3 Deselect all choices except the File Name, Quadrant Label, Percent of Gated,
and Percent of Total; select 1 Column in the Header field (Figure 8-5).
202
BD CellQuest Pro Software User’s Guide
Figure 8-5 Editing Quadrant Statistics
4 Click OK; then resize and reposition the Quadrant Statistics view on the
Experiment document.
Drag on one of the handles to resize the view; drag the
border to move the reduced view to the right of the FL1
vs FL2 contour plot.
The calculated statistics for NORM001 show that most
of the gated events fall in the lower-left (LL) quadrant. In
this example, 98.94% of the gated events are negative for
FL1 and FL2 which would be expected because the subclass control accounts
for nonspecific antibody binding.
5 Select the histogram; choose Histogram Stats from the Stats menu.
The changes you made in the Edit
Quadrant Statistics dialog box also
apply to the histogram statistics.
Position the statistics view to the right
of the histogram.
6 Print the results for NORM001.
Choose Print One from the File menu to print a single copy of the
document.
Chapter 8: Analyzing Data
203
Using the Next Data File Command
The Next Data File and Previous Data File commands in the Plots menu allow you
to select one or more analysis plots and increment or decrement their data files by
any number. The next data file is defined as the next file of the appropriate type in
alphanumeric order within the same folder as the original file. The new data
replaces the original data. Any associated views, such as statistics and annotations,
are also updated with the new information. For example, if the original annotation
was the patient ID, the new annotation is the patient ID from the new file.
The Next Data File command is equivalent to choosing Select File from the File
pop-up menu in the plot Inspector and choosing the file n positions down from
the original file in the list, where n is the file increment.
NOTE The Next Data File, Previous Data File, and Change Data File commands
are not available for overlaid histograms.
1 (Optional) To analyze the tubes in order (ie, Tube 1 from Sample 1 followed
by Tube 1 from Sample 2, etc.), change the File Increment; otherwise, skip
to step 2.
•
Choose File Increment from the Plots menu.
•
Enter the number of the next file to analyze according its position in the
list of files in the folder. For example, the sample files contain four tubes
per sample, so to analyze the tubes in order, you would enter 4.
The Next Data File command analyzes the data files in the following
order: NORM001, NORM005, NORM009.
204
BD CellQuest Pro Software User’s Guide
2 Select the three Analysis plots in your analysis document; then choose Next
Data File (z-]) from the Plots menu.
The plots and statistics views display the data from NORM002 if you are
analyzing with a file increment of 1 or from NORM005 if you specified a
file increment of 4.
3 Print or save the results for the data file; then press z-] to analyze the next
data file.
You can save an electronic copy of each analyzed file as an Experiment
document or as a PDF file that can be opened and viewed on any computer
using Adobe Acrobat Reader. See Saving Experiment Documents as PDFs
on page 228 for more information.
4 Repeat step 3 for the remaining data files you want to analyze.
CellQuest Pro will consecutively analyze each data file in the folder.
•
To return to a previous data file, choose Previous Data File (z-[) from
the Plots menu.
•
To switch to another folder, choose Change Data File (z-D) from the
Plots menu and navigate to the new folder in the Location dialog box
that appears.
5 Save the Experiment document.
To save your document as an acquisition template, see page 73.
Special Considerations
Keep the following in mind when using the Next Data File and Previous Data File
commands.
• Within a plot, Next and Previous Data File will call up the next file in the
folder. However, when more than one plot is selected, each plot can display
data from a different folder. In this case, you can increment data file A from
Chapter 8: Analyzing Data
205
Folder A and data file B from Folder B at the same time using the Next Data
File command.
• Only files of the appropriate type are considered in the increment. Non-FCS
files are not considered. If the current plot is a dot plot, a single-parameter
histogram file is not counted in the increment. Folders within folders are not
considered. This is consistent with manually changing the data file; only
those files appropriate to the specific plot type appear in the Open a Data
File dialog box after clicking Select File.
• Next Data File and Previous Data File are all-or-nothing functions. If, for
some reason, the data file of one plot cannot be incremented, none of the
plots will be incremented. If you reach the end of the list of files in a folder, a
message appears, and the first plot that could not be incremented is selected.
• If the next data file does not contain enough parameters, a message appears.
CellQuest Pro matches on parameter numbers, not parameter names, in
FCS files. A given plot, for example, will remember that its x-axis is showing
parameter 2 and its y-axis is showing parameter 5. No error occurs if
subsequent data files have at least as many parameters as the highest
numbered parameter being displayed.
An example illustrates some of these points. There are two analysis plots in an
Experiment document: a histogram from DATA001, which is located in the Data
Files folder, and a dot plot from NORM001, which is located in the Sample Files
folder.
Applying the Next File command to the histogram is straightforward. All files in
the Data Files folder (Figure 8-6) are appropriate for histograms, so the next file is
easy to identify.
• Set the file increment to 1, and the next file displayed is DATA002.
• Set the increment to 2, and the next file displayed is DATA003, and so on.
206
BD CellQuest Pro Software User’s Guide
Figure 8-6 Data Files folder contents
Applying the Next File command to the dot plot is more complex. Figure 8-7
shows the contents of the Sample Files folder from the Finder.
Figure 8-7 Sample Files folder contents shown from the Finder
Figure 8-8 shows the contents of the Sample Files folder when you choose Select
File from the File pop-up menu in the Dot Plot Inspector. Note that the file lists
are different.
Figure 8-8 Sample Files folder contents for a dot plot
Chapter 8: Analyzing Data
207
• A dot plot cannot be generated from a histogram file, so the Histogram File
is not listed when the dot plot is chosen.
• A ReadMe file is not an FCS data file; thus it is not listed when the dot plot
is chosen.
With this information in mind, you see that selecting the dot plot displaying
NORM001 and choosing Next File with the file increment set to 1 displays
NORM002. If you again choose Next File, Old Folder is ignored. Therefore, the
next file displayed is TEST1. The Previous File command works in the same way,
only moving up, rather than down, the list of files.
Analyzing Groups of Data Files
This section describes how to use CellQuest Pro analysis features to analyze
multiple tubes in a panel or multiple panels. There are several ways to analyze a
series of data files.
• You can use the Next File command to manually advance a series of data files
through the designated analysis template until all files in a folder have been
analyzed, as described in the previous section.
• You can use the Load Sample command to associate a set of plots with a
panel and to display similar data files in each plot, then use the Next Sample
File command to analyze the next data file in the series.
• You can set up batch analysis to automatically advance a series of data files
through an analysis template, to pause between each data file, and to print a
copy of each analysis document before proceeding with the next file.
Using the Load Sample Feature
The Load Sample feature is useful for managing the analysis of large, multi-tube
data files, especially when the files were acquired as part of a panel. The Load
Sample command displays a set of FCS data files in a group of plots in an
Experiment document. In many ways, the Load, Next, and Previous Sample
commands are similar to the Next and Previous Data File commands, with
additional capabilities.
208
BD CellQuest Pro Software User’s Guide
To use the Load Sample feature, you must enter a unique Sample ID or Patient ID
before acquiring samples.
• When loading data files into multiple plots, if the data file for one plot is
missing, the plot will be left blank but all other plots will still be loaded.
• When using Next Sample, you don’t have to know the panel length or
subsequent file increment. Only appropriate data files are loaded into each
plot. For example, a 3/4/45 plot would only load 3/4/45 tube data.
To understand how to use the Load Sample feature, review the following example.
The following IMK Lymph panels have been acquired and will be analyzed using
Next Sample:
• Panel A = Sample 1, tubes 1–6
• Panel B = Sample 2, tubes 1–3, 5, 6 (tube 4 is missing)
• Panel C = Sample 3, tubes 1–8
The Experiment document displays Panel A plots for tubes 1, 2, 4, and 6. These
plots are listed under the appropriate tube names in the Analysis Browser.
You can use the Analysis Browser or the Plots menu to set up the Load Sample
feature to display the next series of data files in the plots. Use Previous Sample to
display the series of data files before the currently selected sample.
Loading Samples Using the Browser
The Load, Next, and Previous Sample commands are available from the pop-up
menu located next to each panel row in the Analysis Browser (Figure 8-9). When
Chapter 8: Analyzing Data
209
you use the Browser commands, the next set of data files will be displayed only in
the plots shown in the Browser panel.
Figure 8-9 Choosing Load Sample from the Analysis Browser pop-up
1 In the Browser, click on the pop-up control next to the panel folder and
choose Next Sample from the pop-up menu.
The plots in the Experiment document automatically display the data files
from Panel B.
Note that the missing data file from Tube 4 (CD3/CD4 tube) has been
skipped but all other plots have still been loaded.
2 Choose Next Sample again from the Browser pop-up menu.
The plots in the Experiment document automatically display the data files
from Panel C.
210
BD CellQuest Pro Software User’s Guide
Now that data is available for the CD3/CD4 tube, the plot is able to display
data and thus reappears in the analysis tube list.
3 Choose Previous Sample from the Browser pop-up menu to display the data
files from Panel B.
Loading Samples Using the Plots Menu
Use the menu commands in the Plots menu if you want to display data in a
selected set of plots rather than a predefined panel.
1 In the Experiment document, select the plots you want to display data.
Select multiple plots by holding down the Shift key while clicking on the
plot borders.
NOTE Selected plots must exist as part of the same panel and sample.
2 Choose Next Sample File from the Plots menu.
The selected plots in the Experiment document automatically display the
next series of data files.
3 Repeat step 2 for the remaining data files you want to analyze.
Chapter 8: Analyzing Data
211
In summary, choosing Next Sample File displays the following data files, using the
panels described in the example on page 209.
Plots
First Data Set
Next Sample
Next Sample
Plot A
Sample 1, Tube 1
Sample 2, Tube 1
Sample 3, Tube 1
Plot B
Sample 1, Tube 2
Sample 2, Tube 2
Sample 3, Tube 2
Plot C
Sample 1, Tube 4
(missing)
Sample 3, Tube 4
Plot D
Sample 1, Tube 6
Sample 2, Tube 6
Sample 3, Tube 6
Changing Directories
Next Sample will continue displaying data files until you reach the end of the file
list in the selected folder. Use Load Sample to switch to another file directory.
NOTE The Analysis Browser and Experiment document should contain a tube list
and plots appropriate for the data files to be displayed.
1 In the Browser, click on the pop-up control next to the panel or tube folder
and choose Load Sample from the pop-up menu that appears.
Alternatively, choose Load Sample Files from the Plots menu. The Load
Sample dialog box appears.
2 Navigate to the data file you want to display; select the data file, and click
Load Sample.
212
BD CellQuest Pro Software User’s Guide
The dialog box lists the first tube for each sample in the current folder. The
panel and tube names are listed in the FCS File Info box.
3 Choose Next Sample File from the pop-up menu or the Plots menu to
continue displaying data files from the same folder.
Setting Up Batch Analysis
Batch analysis can be used to automatically advance a series of data files through an
analysis template, to pause between each data file, and to print a copy of each
analysis document before proceeding with the next file. This feature is based on
the Next Data File command described on page 204; the special considerations
described on page 205 apply.
1 Choose Setup from the Batch menu.
2 Select the Plots and Stats to Process in the Batch Setup dialog box.
•
Select All to process all plots and statistics views in the CellQuest Pro
Experiment document.
•
Select Currently selected to process a subset of plots and statistics views.
3 Select the amount of time to pause after each file increment.
Chapter 8: Analyzing Data
213
•
Select None to process each file in turn without pausing.
•
Select Until manual resume to stop after each file increment until you
tell the computer to proceed.
•
Select For ... seconds to specify a pause time.
4 Select Print after each file increment.
A printout of the analysis is generated for each data file.
5 Select Export statistics.
Specify whether to create a new statistics file or append to an existing file.
See Exporting Statistics on page 216.
6 Specify the File Increment.
Enter the number of the next file to analyze according to its position in the
list of files in the folder.
7 Click OK.
The settings entered in the Batch Setup dialog box are saved with the
CellQuest Pro Experiment document.
Running Batch Analysis
1 Choose Run from the Batch menu (z-M) to begin batch analysis.
A Batch Control floater appears.
stop
pause
resume
214
BD CellQuest Pro Software User’s Guide
2 Use the batch controls if you want to stop or pause batch analysis.
If you selected Pause until manual resume in the Batch Setup dialog box, you
will need to click the resume button after each data file is processed.
Otherwise, batch analysis proceeds automatically unless you click the pause
button. A dialog box appears at the end of the batch run to notify you that
the run is finished.
The Batch Control floater closes automatically when a batch finishes or
when you press the stop button in the floater.
Saving FCS Files
CellQuest Pro automatically saves FCS data files following acquisition. However,
you can use the Save FCS File command to save a subset of the data in a separate
data file without affecting the original data file. There is no reference in the new
FCS file to the original file and the acquisition date of the new file is updated to
reflect the date it was saved. Instrument settings are not saved in these new FCS
files.
To save a data subset:
1 Select a plot of interest.
2 Choose Save FCS File from the File menu.
The type of selected plot determines the information saved.
•
For a dot plot, gated list-mode data containing all the parameters of the
original file is saved.
For example, you can analyze a four-parameter, 10,000-event file and
apply a gate to include the population of interest. Assuming the gate
contains 3,000 events, you can save all four parameters for just those
3,000 events of interest in a new FCS file.
•
For an unsmoothed histogram plot (one histogram), single-parameter
(SP) data containing only the parameter currently displayed in the
Chapter 8: Analyzing Data
215
histogram are saved. You cannot save an SP file from a smoothed
histogram.
If items are waiting to be recalculated, Save FCS File is not enabled. See
Automatic vs Manual Calculation on page 167.
Exporting Statistics
CellQuest Pro allows you to export the statistics generated during data analysis for
use in a spreadsheet, word processing, or other program. Statistics in each statistics
view in the document are exported to a file you specify. There are three ways to
export statistics:
• Choose Export Statistics from the File menu—use this option to export a
selected set of statistics during manual data analysis.
• Choose Append Statistics from the File menu—use this option to add a
selected set of statistics to an existing statistics file during manual data
analysis.
• Select the Export Statistics checkbox in the Batch Setup dialog box—use this
option to export statistics during batch analysis. You can specify whether to
create a new file or append to an existing file. Statistics are automatically
exported after each Next Data File command.
To export a selected set of statistics:
1 Select the statistics view you want to export.
2 Choose Export Statistics from the File menu.
3 Enter a name for the statistics file and specify a storage location in the dialog
box that appears; click Save.
To append a selected set of statistics to an existing statistics file:
1 Select the statistics view you want to export.
216
BD CellQuest Pro Software User’s Guide
2 Choose Append Statistics (z-’) from the File menu.
3 Locate the statistics file in the dialog box that appears; click Append.
To export a batch of statistics:
1 Choose Setup from the Batch menu.
2 Select the Export Statistics checkbox in the Batch Setup dialog box.
3 Select the New File or Existing File button.
•
Click New File to create a new statistics export file. Enter a file name
and specify a location in the dialog box that appears; click Save.
•
Click Existing File to append statistics to an existing file. Navigate to the
statistics file in the location dialog box that appears; click Append.
The Batch Setup dialog box displays the current statistics export file and its
path in the Export File field.
4 Click OK.
Using Quantitative Calibration
Traditional analysis paradigms in flow cytometry include: a) percent positives and
negatives based on an isotype control, and b) absolute counts (cells/µL) using a
known number of reference beads. The continuum of fluorescence staining
intensities from the negative to the brightly positive is often expressed in relative
terms such as dim and bright.
Quantitation, the expression of the staining intensity levels in terms of
fluorophores per cell, can provide a more meaningful classification of data.
Chapter 8: Analyzing Data
217
Quantitation of fluorophores is a necessary first step in the assessment of antigen
density on cells. Other antigen-specific parameters, for example, epitopes per
antigen and epitopes bound per antibody, and variables associated with sample
preparation, such as avidity and concentration, must be controlled as well.
Quantitation can be done using QuantiBRITE™ beads with a known number of
PE molecules for each of four bead populations. By plotting the logarithm of the
geometric means of FL2-H of the four bead populations against the logarithm of
the number of PE molecules per bead, a linear regression can be done on the data
to get the equation:
y = mx + c
where m is the slope of the regression line and c is the y-intercept.
y = 0.99707 x –1.7247
Log FL2
R2 = 1.000
Log PE/bead
This equation is then used to transform FL2-H into the number of PE molecules
for a series of assays. Refer to the QuantiBRITE PE product data sheet for more
information.
Setting Up for Quantitative Calibration
While CellQuest Pro cannot use the regression equation to transform FL2-H, you
can use the Quantitative Calibration command in the Acquire menu to calculate
the regression line and save the slope and intercept information in data files for use
in other applications.
During installation, the Installer places a Quantitation Acquisition document in
the Sample Files folder in the CellQuest Pro application folder. Use this document
to set up quantitative calibration.
218
BD CellQuest Pro Software User’s Guide
1 Launch CellQuest Pro, if it isn’t already open.
2 Open the Quantitation Acquisition document in the Sample Files folder.
The folder can be found in the CellQuest Pro Folder in the BD Applications
folder.
The Quantitation Acquisition document appears.
Notice that the document contains:
•
an acquisition-to-analysis dot plot of FSC-H vs SSC-H with a region for
the bead singlets
•
an acquisition-to-analysis histogram plot of FL2-H gated on bead
singlets
•
a Histogram Statistics view
3 Customize your template.
Add plots to this document that you will use to optimize the cytometer and
to acquire data for your cellular sample.
4 Save your modified Experiment document.
Change the folder location if necessary.
Chapter 8: Analyzing Data
219
5 Place your cellular sample on the sample injection port (SIP) and optimize
the instrument settings; save an instrument settings file.
Using Setup mode, adjust all the parameters for your cellular assay. Make
sure the instrument is properly compensated. See Optimizing the
Instrument for Biological Samples on page 85.
6 Prepare the QuantiBRITE bead sample according to the package insert and
place it on the SIP.
You can change the FSC and SSC parameter settings to gate on bead singlets
without altering quantitation. All instrument settings for fluorescence and
compensation must be the same as the cellular assay settings.
7 Adjust the region in the scatter plot over the singlet bead populations
(Figure 8-10).
Figure 8-10 Gating on bead singlets
8 Uncheck Setup in the Acquisition Control window, and acquire a data file of
10,000 events.
9 Adjust the markers in the histogram over each bead peak (Figure 8-11).
220
BD CellQuest Pro Software User’s Guide
Figure 8-11 FL2-H histogram plot with markers
The statistics will be updated after adjustments have been completed.
10 Choose QuantiQuest from the Acquire menu.
The Quantitative Calibration window appears.
11 Enter the lot-specific values provided on the flyer packaged in the
QuantiBRITE PE kit.
Chapter 8: Analyzing Data
221
12 Select the Histogram Statistics view (FL-2 beads).
13 Click Copy Means on the Quantitative Calibration window.
The geometric means in the selected Histogram Statistics view are copied to
the Quantitative Calibration window.
The Copy Means button is enabled only when you have selected one FL2-H
Histogram Statistics view and that statistics view displays all four of the
following:
•
analysis statistics, not live statistics
•
the geometric mean column
•
exactly four markers, not including the All marker
•
Log Data Units set to Linear Values
When you click Copy Means, the geometric means for the four markers in
the view are sorted and copied in to the Geometric Mean column of the
Quantitative Calibration window. The statistics data file name is also
displayed.
14 Click Calibrate.
CellQuest Pro performs the regression analysis and displays the results in the
Slope, Intercept, and R-Squared fields as illustrated in Figure 8-12.
222
BD CellQuest Pro Software User’s Guide
]
Figure 8-12 Results displayed in Quantitative Calibration window
NOTE Once you click Calibrate, do not click the Copy Means button again
or change a PE molecule value. If you do either, the results are presented as
***, or undefined values, and you will have to redo the calibration.
You can print the active Quantitative Calibration window. Choose Print
from the File menu.
15 Save your Experiment document.
When you save an Experiment document used to perform quantitative
calibration, the PE molecules per bead values you entered in the
Quantitative Calibration window are saved; the mean and regression results
are not saved, however. You must recalculate to get the calibration
information when you reopen the document for further data file acquisition.
If the Quantitative Calibration window contains defined values when the
calibration process is complete, the quantitation information is saved in all
subsequently acquired data files.
16 Acquire your cellular samples.
You must use the same instrument settings and the same Experiment
document.
Chapter 8: Analyzing Data
223
Data files saved using this document will contain new keywords in the text
sections which define the results of the calibration. If you use a different
document to collect your data files, the keywords will not be present in the
file text section.
224
BD CellQuest Pro Software User’s Guide
9
Publishing and Presenting Data
• exporting plots
• saving Experiment documents as PDF files
225
After aquiring and analyzing data in CellQuest Pro, you can save plots as graphical
objects to export into a word-processing or graphics program, send by electronic
mail, or publish on a website. With the addition of the Adobe PDFWriter, you can
save entire Experiment documents as Adobe PDF (portable document format)
files. PDF files can be opened and viewed on any computer using Adobe’s free
Acrobat Reader software.
Before Beginning This Chapter
You should:
• be familiar with Experiment document components and views. Review
Chapter 2.
• have an analyzed data file with plots available for export. Review Chapter 8.
Exporting Plots
CellQuest Pro can export plots in the Experiment document view area in a variety
of graphical formats. Apple Quicktime 4.0 or later is required to support all
formats. You can also copy plots from an Experiment document and paste them
into any graphics or word processing application. When pasting plots, the
background automatically defaults to white.
1 Select the plot you want to export.
2 Choose Export Graphic from the File menu.
3 Select the format of the exported graphic file from the dialog box that
appears (Figure 9-1).
You can choose from:
226
•
JPEG—retains all color information in an RGB image
•
GIF—minimizes file transfer in compressed format
BD CellQuest Pro Software User’s Guide
•
BMP—serves as standard bitmap image
•
PNG—developed as alternative to GIF format
•
PICT—compresses images having large areas of solid color
Figure 9-1 Export graphics dialog box
4 Select the resolution.
Choose from 72 (default), 150, or 300 dpi. A higher resolution results in a
higher quality graphical output, but a larger file size.
5 Choose the background color of the exported plot object.
Specify a white or black background. If your plot uses light-colored dots, you
might want to pick a black background or change the color of the dots using
the Inspector.
6 Enter a file name and navigate to a location; click Save.
Helpful Hints for Publication-Quality Graphics
• For presentations, use the Highlite Dots feature to enlarge the events of
interest if you will be showing them on a slide. See Highlighting Dots on
page 162.
Chapter 9: Publishing and Presenting Data
227
• Use the Inspector to format the text style of the plot to your satisfaction
before exporting.
• Use the Inspector to hide or show the plot title.
• If you want to change the plot, export it to a graphics program and make any
changes there.
Saving Experiment Documents as PDFs
With the addition of the Adobe PDFWriter, CellQuest Pro Experiment
documents can be saved as Adobe PDF files that can be viewed on any computer
using Adobe’s free Acrobat Reader software.
The PDF version of a CellQuest Pro document contains all of the items in the
view area of the Experiment document (plots, statistics, expressions, annotations,
etc). It does not include the contents of any CellQuest Pro window such as the
Inspector or the Browser.
Use the following method to save an Experiment document as a PDF file. To avoid
having to select the Acrobat PDFWriter as the default printer, create a shortcut as
described on page 229.
1 Select Chooser from the Apple menu.
2 Select Acrobat™ PDFWriter as the printer; close the Chooser.
3 Print the Experiment document by choosing Print (z-P) from the File
menu.
4 To view the PDF file after it is created, check the View PDF File checkbox.
228
BD CellQuest Pro Software User’s Guide
5 Click OK.
6 Enter the name and navigate to a file location in the dialog box that appears;
click Save.
NOTE Retain the .pdf suffix in the filename to open the file on multiple
computer platforms.
Creating a PDFWriter Shortcut
To avoid having to select the Acrobat PDFWriter as the default printer, create a
shortcut.
1 Choose Control Panels from the Apple menu.
2 Select PDFWriter Shortcut from the drop-down menu that appears to the
right.
3 Follow the instructions in the dialog box to create a shortcut.
You can specify which key combination to use for the shortcut.
Chapter 9: Publishing and Presenting Data
229
230
BD CellQuest Pro Software User’s Guide
Glossary
analysis
The software function of numerically and graphically
manipulating data to generate statistics.
annotation
A label or comment read from a data file and displayed in the
Experiment document.
coefficient of variation
(CV)
The standard deviation of the data divided by the mean of the
data; typically expressed as a percentage. When applied to
channel data measured on a population of cells, the CV is a
measure of variation independent of the population mean.
contour plot
A graphical presentation of two-parameter data in which
contour lines show the distribution of events. Similar to a
topographical map, the contour lines show event frequencies as
peaks and valleys.
data file
A collection of measured values from a single sample combined
with text describing the sample that has been stored to disk. See
also list-mode file.
dot plot
A graphical means of representing two-parameter data. Each
axis of the plot displays values of one parameter. A dot
represents an event (particle).
Experiment document
A collection of gates, plots, regions, statistics, and text, defined
by the user.
Experiment window
The window for viewing parts of an Experiment document.
FCS
See Flow cytometry standard.
Flow cytometry standard
Standard format for flow cytometer data files.
231
gate
A combination of one or more previously defined regions that
define a subset of the total sample population. The logical
operators AND, OR, and NOT, along with parentheses, if
necessary, are used to define logical gates.
histogram
A graphical means of presenting single-parameter data. The
horizontal axis of the graph represents the increasing signal
intensity of the parameter, and the vertical axis represents the
number of events (count).
list-mode file
An unprocessed data file containing all the measured parameters
for each particle in the sample, as well as information describing
the sample.
marker
A lower and upper boundary on a histogram that allows you to
obtain statistics for a particular section of a histogram.
mean channel
The average channel number of a sample from a population of
cells.
parameter
A measurement of a cell property that is ascertained as the cell
passes through the laser beam. Each parameter is the output of a
single photomultiplier tube or photodiode, measuring
fluorescent or scattered light.
peak
The number of events in the peak channel.
peak channel
The channel number containing the maximum number of
events in a marker.
region
A boundary drawn around a subpopulation to isolate events for
analysis. Regions can be combined to create gates.
single parameter (SP) file
A file that contains event counts for one parameter.
threshold
A trigger signal and level of discrimination to eliminate
unwanted events. Only events with parameter values above the
threshold will be analyzed.
tool palette
A collection of tools used for creating plots, markers, regions,
free text, and recalculation.
232
BD CellQuest Pro Software User’s Guide
Appendix A
Keyboard Shortcuts
233
Command-key equivalents, also known as keyboard shortcuts, are combinations of
key presses that you can use instead of mouse actions to perform a task. They are
convenient alternatives to using the mouse to choose menu commands. They
appear in the pull-down menus next to their menu items.
From the File menu
To open a new Experiment document
z-N
To open a saved Experiment document
z-O
To close the active Experiment document
z-W
To save the active Experiment document
z-S
To append statistics to an exported file
z-'
To print the active Experiment document
z-P
To quit CellQuest Pro
z-Q
From the Edit menu
To undo the most recent change
z-Z
To cut an item to the Clipboard
z-X
To copy an item to the Clipboard
z-C
To paste an item from the Clipboard
z-V
To select all items in the active document or view
z-A
To access the Text Settings dialog box
z-T
From the Cytometer menu
234
To open the Detectors/Amps window
z-1
To open the Threshold window
z-2
To open the Compensation window
z-3
To open the Status window
z-4
BD CellQuest Pro Software User’s Guide
From the Plots menu
To reformat the selected plot
z-F
To change the data file for the selected plots
z-D
To get the next data file for the selected plots
z-]
To get the previous data file for the selected plots
z-[
From the Gates menu
To open the Gate List window
z-G
To recalculate any data in the Experiment document
z-=
From the Batch menu
Run
z-M
From the Acquire menu
To connect/disconnect the cytometer
z-B
Appendix A: Keyboard Shortcuts
235
236
BD CellQuest Pro Software User’s Guide
Index
Numerics
3D plot
Color Palette, using 42–43
creating 28
defined 52
formatting 62–63
3D Plot Inspector 62
A
Acquire menu commands 235
acquisition
controls 91
displaying live statistics 132
ending 136
Experiment document setup 86–89
gate 115, 117
monitoring 135
of data 133–134
pausing 136
plots 50
pointer 114, 135
status icon 114
time limit 118
Tube List 114
tube list setup 125–126
tube order 126
using counters 130–132
Acquisition & Storage Inspector 129
Acquisition Browser 112–114, 133
acquisition-to-analysis plots 50
active objects 30–31
Adobe
Acrobat 16, 17
PDF 16, 228
Amp Gain, adjusting 82, 90–91
analysis
batch 208–215
Experiment document setup 199–208
Next Data File 204–208
plots 50, 200–202
Previous Data File 204–208
Sample Files, using 198–199
statistics, displaying 202–203
Analysis Browser 196–198
Annotation Inspector 39
annotations 37–39
Apple QuickTime 15, 226
Arrow tool 30
assistance, technical xi
Auto Recalculate 167–168
AutoSORT window 107
B
background, plot image 227
banners, adding 43–45
Basic Plot Inspector 52, 53
batch analysis 208–215
Batch menu commands 235
BD Applications folder 18
BD Files folder 18
237
BD Inits 15, 19–20
BDPAC 102–104
beads, CaliBRITE 78
BMP file format 227
Browser
acquisition setup controls 113
Acquisition view 112–114
Analysis view 196–198
entering comments 124
setting up tube list 125–126
C
Calculator tool 30, 168
Calib file, restoring settings 88–89
calibration, quantitative 217–224
setting up for 218
See also quantitative calibration.
CaliBRITE beads 78
CellQuest Pro software
batch analysis 208–215
file compatibility 15–16
file types 24–25
graphics, exporting 226–227
INITs, interference of 15
installing 17–18
launching 20–21
limitations xi
modem, using with 14
quitting 21
registering 21
requirements, hardware 14
requirements, software 15
using for sorting 105–109
channel values 80, 168–169
threshold 83
CHRONYS software 15
238
BD CellQuest Pro Software User’s Guide
CloneCyt Plus software 16, 57
collection criteria 117
Color Palette 42–43
Command-key equivalents x, 234–235
comments, entering 113, 124
compatibility, other software 16
compensation, adjusting 84, 96–99
CONSORT 30 software 15
CONSORT File Exchange 15
contour plot
defined 51
formatting 58–60
Contour Plot Inspector 58
Contour-Plot tool 28
controls
compensation 85
instrument 79–84
subclass 85
conventions
file naming 25–26
text x
coordinates, cursor 27
counters 130–132
cursor coordinates 27
custom keywords, defining 141–142
cytometer
connecting to 79
controls 79–84
printing settings 101
quality control 78
restoring Calib settings 88–89
restoring settings 100–101
saving settings 100
starting 86
status, checking 101–104
Cytometer menu commands 234
D
E
data
acquisition 133–134
analysis See analysis.
batch analysis 208–215
exporting 216–217
Sample Files, using 198–199
saving 75, 215–216
data files
compatibility 15–16
FCS format 15
HP files, converting 15
saving 75
density plot
defined 51
formatting 60–62
Density Plot Inspector 60
Density-Plot tool 28
deselected objects 32
detectors
adjusting fluorescence 93–95
signal 80
Detectors/Amps, adjusting 80–82
document size 36–37
dot plot
creating gated 93–95
defined 50
formatting 56–57
multicolor gates 160–162
Dot Plot Inspector 56
Dot-Plot tool 28
Duplicate option 52, 55
Edit menu commands 234
Elliptical-Region tool 29, 145
Eve Init 18
Experiment document
acquisition setup 86–89
analysis setup 199–208
banners, adding 43–45
components 26–27
customizing 36–47
files 24
grids, displaying 45–46
PDF files, saving as 228–229
resizing 36–37
saving 73–74
Snap to Grid 46–47
templates 73–75
exporting plots 226–227
Expression Editor 191–193
tool 30
expressions
creating custom 191–193
editing 194
F
FACS Loader 14
FACSCalibur 105–106
FACScan Research software 15
FACSComp 78
FACSConvert software 15
FACSNet Mac 15
FACSort 105–106
FACStar Plus Research software 15
FACStation 14
Index
239
FACSVantage SE 16, 106–109
FCS files 15, 24, 75, 215–216
FCS Keyword Inspector 52
file
CellQuest Pro 24–25
count 124
data files 24
Experiment document 24
instrument settings 25
name, defining 122–124
naming conventions 25–26
prefix name 124
sharing 133–??
statistics 25
storage location, defining 122–124
suffix name 124
File menu commands 234
flow cytometer compatibility 14
Flow Cytometry Standard format See FCS files.
fluorescence, background 93
G
Gate List 159–160
gated plots, creating 93–95
gates
combined regions 157–159
copying 160
defined 144
defining 157–160
Gate List setup 162
multicolor 87, 160–162
setting 91–92
statistics 182, 188
storage 119
Gates menu commands 235
geometric mean 170
Geometry Inspector 35, 52
GIF file format 226
Global Acquisition & Storage settings 113,
115–120
graphics, exporting 226–228
grids
displaying 45–46
units, specifying for 45
H
hardware requirements 14
highlighting dots 162, 227
Hilite feature 162, 227
histogram plot
Color Palette, using 42–43
creating regions 144–145
defined 52
displaying statistics 175–176
formatting 65–66
legends 70
markers 171–172
overlays 66–70
statistics 189
using tools for 70–72
Histogram-Marker tool 28
Histogram-Plot tool 28
Histogram-Region tool 29
HP files, converting 15
I
Inits, BD 15, 19–20
Index
240
Inspectors
3D Plot 62
Acquisition & Storage 129
Annotation 39
Basic Plot 52
Contour Plot 58
Density Plot 60
Dot Plot 56
editing tubes 126–127
FCS Keyword 52
Geometry 35, 52
Instrument Settings 130
Stats 190
Text Style options 32–34
window contents 33
instrument
controls 79–84
printing settings 101
quality control 78
restoring Calib settings 88–89
starting 86
status 101–104
instrument settings
adjusting for sorting 105–109
printing 101
restoring 100–101
saving 100
Instrument Settings Inspector 130
J
JPEG file format 226
K
L
labels, parameter 124–125
launching software See CellQuest Pro software.
legends, histogram 70
linear
amplification 82
values 168–169
LoaderManager software 14
log amplification 82
Log Data Units 168–169
lymphocytes, gating 91–92
LYSYS II software 15
M
Mac OS 15
Marker List 172–173, 174
Marker tools 28
markers
converting to regions 174
copying 174
histogram 171–172
list of 172–173
quadrant 93, 181
means, calculating 170
menu bar 26, 27
monitoring acquisition 135
multicolor gating 87, 160–162
MultiSET software 15
N
Next Data File 204–208
keyboard shortcuts See Command-key
equivalents.
keywords, defining 141–142
Kilmogorov-Smirnov See K-S statistics.
K-S statistics 179–180
Index
241
O
objects in view area
active 30–31
aligning 35
deselected 32
resizing 35
selected 31
optimization controls 85
overlaid histogram
defined 66
statistics 176–179
P
palette tools 28–30
panels
creating 138–140
editing 140–141
parameters
labels 124–125
resolution 119
saving 119, 120–122
using time 64–65
patient ID 113, 124
PDF files 16, 228–229
percent, gated vs. total 169
photodiode 80–81
photomultiplier tubes See PMTs.
PICT file format 227
plots
acquisition 50
acquisition-to-analysis 50
aligning 55
analysis 50, 200–201
associating with tubes 127–128
creating 52–55
deleting 55
Duplicate option 52
exporting 226–227
formatting 56–72
gates 115
magnifying 29
tools, creating with 28–30, 53
Plots menu commands 235
PMTs
defined 82
FACSComp setup 78
PNG file format 227
Polygonal-Region tool 29, 145
portable document format See PDF.
Previous Data File, using 204–208
ProCOUNT software 15
pulses, test 104
Q
quadrant
markers 93, 181
statistics 94, 182, 187
Quadrant-Marker tool 28
quality control, instrument 78
QuantiBRITE beads 218
quantitative calibration 217–224
Quantitation Acquisition document 218
quantitation, defined 217
Quantitative Calibration window 218,
222, 223
setting up for 218
Index
242
R
ReadMe file ix, 17
Reagent List 137
recalculation, automatic vs. manual 167–168
Rectangular-Region tool 29, 145
Region List 154–155
regions
combined in gate definitions 157–159
copying 155–157
creating 144–148
defined 144
deleting 152
displaying hidden 153
editing 150–151
hiding 153
histogram 144–145
naming 154–155
polygonal 146–147
Region List, using 154–155
rotating 151–152
Snap-to 148–149
statistics 182, 187
registering software See CellQuest Pro
software.
requirements
hardware 14
software 15
resolution
parameter 119
plot image 227
ribbon 27
S
Sample Files folder 18
sample ID 113, 124
saving
data 75, 215–216
Experiment documents 73–74, 228–229
security module 14
installing 17
selecting objects 31
Selection tool 28
settings
Global Acquisition & Storage 113, 115–
120
tube-specific 128–130
setup mode, acquisition 91, 113
signal detectors 80
SimulSET software 15
size box 27
Snap to Grid 46–47
Snap-To regions
copying 149
creating 148–149
reading new data into 149
Snap-to regions
tool 29
software compatibility 16
sorting 105–109
statistics
Auto Recalculate 167–168
displaying analysis 202–203
displaying live 132
editing 184–185
exporting 216–217
files 25
formatting 190
gate 166, 182, 188
histogram 166, 175–176, 189
K-S 166, 179–180
means 170
overlaid histogram 176–179
percent gated vs. total 169
quadrant 94, 166, 182, 187
region 166, 182, 187
Stats Inspector 190
Status window 104
storage gate 115, 119
Index
243
T
V
technical assistance xi
templates, Experiment document 73–75
test pulses 104
text 37–41
aligning 34, 41
background 34, 41
Color Palette, using 42–43
formatting 32–34
free, adding 39–40
Inspector options, using 32–34
settings 40–41
tool 30, 39, 40
Text tool 30, 39, 40
threshold, adjusting 83, 90–91
time
limit 118
parameter 64–65, 119
resolution 118
tool palette 26, 27, 28–30
tools
Arrow 30
Calculator 30, 168
Expression Editor 30
Marker 28
Plots 28
Regions 29
Selection 28
Text 30
using histogram 70–72
Zoom 29
Tube Inspector 126–127
tubes, PMT defined 82
tube-specific settings 112, 114, 128–130
values, channel 80, 83
voltage, adjusting 81–82, 90–91, 95–96
W
Windows menu 196
WorklistManager software 16
X
X and Y parameters, changing in plots 63–64
Z
zoom box 26, 27
Zoom tools 29
U
unit settings 45
Universal Serial Bus See security module.
Index
244
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertising