Guava easyCyte System User Guide

guava easyCyte™ System
User Guide
For systems running guavaSoft™ software, version 3.1
For Research Use Only; not for use in diagnostic procedures
0110-8493 Rev B
Copyright
Disclaimer
Limitations
Trademarks
Patents
© 2014 EMD Millipore Corporation, Inc. All rights reserved.
No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval
systems, or translated into any form, or by any means: electronic, mechanical, magnetic,
optical, or otherwise, without the prior written permission of EMD Millipore Corporation, 25801
Industrial Blvd., Hayward, CA 94545, United States of America.
EMD Millipore reserves the right to change its products and services at any time to incorporate
the latest technological developments. This guide is subject to change without notice.
EMD Millipore has thoroughly tested the operation of guavaSoft™ Software on Microsoft®
Windows® XP and Windows® 7 operating systems, but does not warrant that the software
functions correctly on any other operating system.
EMD Millipore has not validated the analysis of guava easyCyte™ System data using thirdparty programs and cannot warrant that the results using these programs will be correct.
EMD Millipore does not provide support for any third-party programs.
The guava easyCyte™ System is for research use only; not for use in diagnostic procedures.
The results of the assays are dependent upon the proper use of the reagents and instrument.
Please refer to the appropriate package insert for specific instructions and limitations.
The M logo is a trademark of Merck KGaA, Darmstadt, Germany.
Millipore, FlowCellect, InCyte, easyCheck, guavaSoft, ViaCount, Nexin, and guava are
registered trademarks or trademarks of EMD Millipore Corporation.
Microsoft, Windows, Internet Explorer, and Excel are registered trademarks of Microsoft
Corporation.
Dell is a trademark of the Dell Computer Corporation.
All other trademarks are property of their respective companies.
The guava easyCyte™ System is the subject of issued and pending US patents and foreign
equivalents, including the following US patents:
5,798,222 – Apparatus for monitoring substances in organisms
6,403,378 – Cell Viability assay reagents
6,710,871 and 6,816,257 – Method and apparatus for detecting microparticles in fluid samples
7,320,775 – Exchangeable flow cell assembly with a suspended capillary
7,410,809 – Particle or cell analyzer and method
8,184,271 and 7,847,923 – Differentiation of flow cytometry pulses and applications
0110-8493 Rev B
December 8, 2014
Table of Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
Chapter 1: Introduction
guava easyCyte™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-1
System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2
Hardware Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2
easyCyte™ System Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-4
easyCyte™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-6
guavaSoft™ Software Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-8
Main Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-8
Unlocking guava InCyte™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-10
FlowCellect® Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-11
guava InCyte™ Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-12
About guava InCyte™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-12
Tool Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-13
InCyte™ Acquisition Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-14
InCyte™ Analysis Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-18
Exiting the Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-19
Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-20
Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-22
Chapter 2: Getting Started
System Startup and Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1
Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1
Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2
Quitting guavaSoft™ Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2
Running the easyCheck™ Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
easyCheck™ Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4
Viewing and Exporting easyCheck™ Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5
Viewing Trend Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7
easyCheck™ Procedure Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-8
Chapter 3: Cleaning and Maintenance
Running Quick Clean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1
Running Guava® Clean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2
Backflushing the Fluid System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
Filling the Cleaning Solution Vial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4
iii
Emptying the Waste Vial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5
Cleaning the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6
About the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6
Using the Syringe Tool to Clean the Flow Cell Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6
Replacing the Flow Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-10
Replacing the Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-12
Returning the System for Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-13
Chapter 4: guava InCyte™ Assay
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-1
InCyte™ Application Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2
Plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2
Pie Legend . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-5
guava InCyte™ Sample Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-6
guava InCyte™ Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-13
The Analysis Panes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-14
Analyzing Files Acquired Using InCyte™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-15
Analyzing Files Acquire Using Other guava® Software Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-17
Regions, Gates, and Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-19
Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-19
Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-22
Stat Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-26
Exporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-33
Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-34
Performing Manual Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-34
Performing Semi-automated Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-36
HeatMap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-38
Creating a HeatMap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-39
Thresholds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-43
Metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-43
IC-50/EC-50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-44
Creating an EC-50 or IC-50 Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-45
Special Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-46
Creating a Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-46
Data Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-48
Overlaying Histograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-49
Printing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-50
guava InCyte™ Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-51
Appendix A: Administrator Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Setting up Access Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-1
Administration Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .A-8
Appendix B: Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
Appendix C: Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1
iv
guava easyCyte™ System User Guide
Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
guava easyCyte™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
easyCyte Lasers and Fluorescent Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fluorochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Notice to Purchaser: Limited Use License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
3
4
5
5
6
7
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
v
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guava easyCyte™ System User Guide
Preface
The guava easyCyte™ System is an automated, desktop cell analysis system that can
perform a wide range of multi-color cellular and assays. The system is equipped with
three lasers—violet, blue, and red—allowing you to analyze up to 10 fluorescence
parameters, as well as forward and side scatter. It accommodates individual
microcentrifuge sample tubes.
guavaSoft™ software, used to set up and maintain the system, offers a number of assayspecific acquisition and analysis modules, depending on your applications. Use the guava
InCyte™ software module to acquire and analyze data. InCyte™ is an open software
package, which you can also use to analyze FCS 3.0 data files acquired from any guava®
software module. For a list of assay-specific software modules, see “Program Search List”
on page 1- 9.
guava InCyte™ application templates are available for all FlowCellect® assays. The
templates provide the plots, parameters, analysis tools (Methods), and statistics for each
application. For more information on these templates, refer to the specific reagent kit user
guide.
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About This Guide
The guava easyCyte™ System User Guide provides detailed information on operating
and maintaining the guava easyCyte™ System, and using the guava InCyte™ software
module for data acquisition and analysis. This guide is intended for the guava easyCyte™
5, 6-2L, 8, and 12 instruments. Any reference to NIR, Red2, and NIR2 parameters, may
not apply to your specific instrument. Refer to “easyCyte Lasers and Fluorescent Filters”
in Specification for a complete list of the parameters available with each system. For
information on using the other guava® software modules, refer to the guavaSoft™
Modules User Guide.
This guide assumes you have a working knowledge of Microsoft® Windows® operating
system. If you have any questions regarding the Dell™ computer or the operating system,
refer to the appropriate manufacturer’s documentation.
For information on preparing samples, refer to the appropriate reagent kit user guide that
was shipped with your reagents.
Conventions Used in This Guide
■ NOTE: Points out additional information that may be helpful.
◆ WARNING: Alerts you to situations that could result in bodily harm, instrument
damage, failure in a procedure, or incorrect results.
Bold: Indicates buttons to click or options within the software to select.
Italics: Used for names of user guides and package inserts, as well as messages that
appear on the screen.
Help
1
Read through the section of the guide specific to the operation you are performing.
Refer to the table of contents and index to locate information. A glossary is included to
assist you with any unfamiliar terms.
2
See the Troubleshooting section in each chapter for a list of problems and suggested
solutions.
3
Refer to the technical support contact information listed below:
• For ordering information or technical support, call toll-free:
USA and Canada, Phone: +1 (800) 645-5476
Fax: +1 (951) 676-9209
• For additional contact information, visit www.millipore.com/flowcytometry
Safety
The guava easyCyte™ System is equipped with safety features for your protection. Use
the system only as directed in this user guide. Do not perform instrument maintenance or
service except as specifically stated. Please read the following safety information before
using the system.
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guava easyCyte™ System User Guide
General Safety
◆ WARNING: If this instrument is not used in the manner indicated by the instructions in
this guide, the safety features of the instrument may be impaired. Follow these
guidelines:
• The use of tubes other than those specified may result in damage to the
instrument.
• Do not run any other programs, including Internet Explorer®, on the laptop while
using guavaSoft™ Software to acquire data. guavaSoft™ Software requires the full
resources of your laptop during data acquisition. Running other programs during a
run may interfere with acquisition or interrupt the run.
Biological Safety
◆ WARNING: All biological specimens and materials that come into contact with them
can transmit potentially fatal disease. To prevent exposure to biohazardous agents,
follow these guidelines:
• Handle all biological specimens and materials as if capable of transmitting
infection. Dispose of waste using proper precautions and in accordance with local
regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear,
and gloves.
• Add 10 mL of bleach to the waste vial after emptying it. Dispose of waste in
accordance with federal, state, and local regulations.
Electrical Safety
The instrument must be connected per the instructions in the user guide. The power
conditioner is required to meet electrical compliance.
◆ WARNING: Turn off the main power switch at the back of the instrument and
disconnect the power cord before replacing fuses.
Laser Safety
The following lasers are used in the easyCyte™ System:
Laser
Wavelength (nm)
Maximum Power (mW)
Violet
405 ±5
110
Blue
488 ±5
160
Red
642 ±5
110
■ NOTE: These are the maximum powers for each laser. For the actual power for a
specified instrument model, see “easyCyte Lasers and Fluorescent Filters” under
Specifications.
ix
No radiation is accessible to the user during normal operation and maintenance. Light
shields within the instrument enclose the path of laser radiation. Additionally, the
instrument enclosure provides secondary protection from any laser radiation.
◆ WARNING: To avoid exposure to laser radiation or electric shock, follow these
guidelines:
• Do not open the instrument or attempt to perform any internal maintenance. There
are no user-serviceable parts.
• Turn off the power to the guava easyCyte™ System before attempting to remove
the flow cell.
◆ WARNING: The use of controls or adjustments or performance of procedures other
than those specified herein may result in hazardous radiation exposure
Labels
Following are examples of the labels affixed to the guava easyCyte™ instrument:
NOTE: The following caution label is visible when
the flow cell access panel is removed. The panel
is to be removed when the system is powered
off. Even when powered on, the accessible laser
radiation falls within Class I limits.
CAUTION - CLASS 3B
LASER RADIATION
WHEN OPEN. AVOID
EXPOSURE TO BEAM
S/N : 8470120101
Passed
By:
D.C.
Manufactured: 04JUN2014
Date: 7/08/05
FUSE
B
USA and Japan
Europe
110 VAC
220 VAC
2A Time-Lag
1.6A Time-Lag
Limitations
• The guava easyCyte™ System is for research use only; not for use in diagnostic
procedures.
• The results of the assays are dependent upon the proper use of the reagents and
instrument. Refer to the appropriate reagent kit user guide for specific instructions and
limitations.
x
guava easyCyte™ System User Guide
CHAPTER 1
Introduction
guava easyCyte™ System
The guava easyCyte™ System streamlines cell phenotyping, drug discovery, and cell
culture monitoring and screening by providing turnkey assays for a wide range of cellbased applications. The system includes the easyCyte™ instrument, a laptop computer
with pre-installed software for data acquisition and analysis, and optimized reagents and
protocols.
guavaSoft™ software includes dedicated application modules for cell- and bead-based
assays, minimizing training requirements. In addition, the software includes the guava
easyCheck™ module, which verifies that the system is performing optimally, and Guava
Clean, a cleaning module that allows you to clean the instrument’s fluid system prior to
shutdown.
guava easyCyte™ System
guavaSoft™ software automatically saves data files, which can be recalled later for offline
analysis. In addition, results are exported to a spreadsheet file.
guava easyCyte™ System
1-1
System Components
The easyCyte™ System is shipped with the following components.
laptop
computer
guava easyCyte™ System
laptop power supply
and power cord
power
conditioner
extension cable
sample tube
holders
power cable
USB cable
guavaSoft™ software CD
Hardware Connections
Although the easyCyte™ System is a portable unit, it contains precisely aligned optical
components that are sensitive to jarring movements. Place the instrument on a stable
surface in a dedicated location in the laboratory. Allow at least 4 inches between the back
of the instrument and the wall for proper ventilation. Maintain easy access to the power
cord in case the instrument needs to be disconnected in an emergency. The initial
installation will be performed by an EMD Millipore field service representative.
◆ WARNING: To avoid damage to the instrument, be sure to remove the shipping
restraint before plugging in the instrument.
■ NOTE: If the instrument needs to be moved to a new location in the lab area or
building, always use two people to lift and a sturdy transport such as a cart. If a longer
distance move requiring packup is required, contact EMD Millipore. A Relocation and
Installation service is available for a fee. See Appendix C: "Ordering Information" for
details.
1-2
1
Connect the cytometer to the laptop with the USB cable (see Figure on page 1-3).
2
Connect the power cable between the power conditioner and a grounded (threeprong) AC power outlet.
guava easyCyte™ System User Guide
3
Connect the extension cable between one of the four power outlets on the back of the
power conditioner and the power input on the cytometer.
■ NOTE: The power conditioner is required to meet electrical compliance.
4
Connect the laptop power supply to the laptop. Plug the power supply into the power
cord, then plug the power cord into the power conditioner.
◆ WARNING: The power conditioner is not a continuous power supply. Ensure that the
instrument is powered on during acquisition.
Connecting the guava easyCyte™ System
5
You can connect the laptop to a local network or the internet. Contact your network
administrator for assistance. You can also connect a printer. If you connect a printer,
you must install the appropriate print drivers.
6
The easyCyte™ System comes with two sets of sample tube holders: a set of holders
for 1.5-mL microcentrifuge tubes and a set of holders for titer tubes. Select the set you
want to use and install them by screwing each holder onto the loader assembly as
illustrated below.
Installing and changing the tube holders
The easyCyte™ System supports the following tubes:
• 1.2-mL microtiter tube with conical tip
• 1.5-mL microcentrifuge tube with conical tip and screw cap (if snap-cap tubes are
used, cut off the cap)
Hardware Connections
1-3
◆ WARNING: The use of tubes other than those specified may result in damage to the
instrument.
7
Refer to “System Startup and Shutdown” on page 2-1 for the correct procedure to
start the laptop computer, easyCyte™ instrument, and guavaSoft™ software.
8
Prime the fluid system. Follow the instructions in “Running Guava® Clean” on page 3-2
using deionized water in place of cleaning solution. Keep the tube of water loaded on
the system whether you are ready to run samples or are going to shut down the system.
easyCyte™ System Troubleshooting
Problem
Possible Causes
Laptop prompts for user ID Laptop is set up for
or password.
authorization.
Solutions
Do not enter password. Click OK
or Cancel to continue. Contact
your IT department for assistance
with any modifications. The
original laptop setup does not
require a password.
During start-up, laptop
freezes on particular
screen.
System may be searching Press Enter to continue. Reboot
for directory during
system, if necessary.
startup.
Message:
The instrument could not
be detected.
Communication problem.
Press Enter to continue. Reboot
system, if necessary. If message
appears after rebooting, contact
EMD Millipore Technical Support.
Message:
1. guava easyCyte™
1. Ensure easyCyte™ System
The instrument appears to
System is not turned on
power cord is properly plugged
be either off or not
or is not getting power.
in and system is turned on.
connected. You can run in 2. Cable connection
2. Ensure USB cable is securely
Analysis mode only.
between easyCyte™
connected to laptop. Use USB
System and laptop is
2.0 ports if possible. Reboot
loose.
computer, if necessary.
3. easyCyte™ System and 3. Turn off easyCyte™ System,
laptop were not
exit guavaSoft™ software,
powered on in correct
restart laptop, turn on
sequence or have lost
easyCyte™ System, wait for
communication.
indicator light before starting the
software.
guavaSoft™ software
Registration code not
launches, but only
entered or not entered
Analysis mode is available correctly.
when an assay is
launched.
1-4
guava easyCyte™ System User Guide
Enter registration code and
ensure all characters are correct.
Problem
Possible Causes
Solutions
Instrument will not power
on.
Loose power cable.
Ensure instrument is plugged.
Check all power connections.
Laptop keeps shutting
down.
1. Power supply to laptop
is faulty.
1. Ensure laptop is plugged in
correctly. Use surge protector
and ensure it is plugged in and
turned on.
2. Adjust power scheme screen
saver options. Click Start>
Settings>Control Panel.
Double-click Display, select
Screen Saver tab, click Settings
under Energy saving features of
monitor. Make sure “Setting for
Always On power scheme” are
all set to Never.
Laptop should not be allowed to
“sleep.” guavaSoft™ software
will stop acquiring data until the
laptop is woken up.
3. Ensure cooling vents are clean
and unobstructed.
2. Screen saver is
interfering.
3. Laptop overheating.
For InCyte™ only
Message:
Sorry - this unlock is
invalid.
Incorrect or no unlock key. Ensure the correct unlock key is
entered. If necessary, contact
EMD Millipore Technical Support
to obtain unlock key.
◆ WARNING: Do not run Excel® software, Internet Explorer® browser, or any other
program on the laptop while using guavaSoft™ software to acquire data from the
guava easyCyte™ System. guavaSoft™ software requires the full resources of your
laptop during data acquisition. Running other programs (even if you are not actively
using them) during a run may interfere with acquisition or interrupt the run.
easyCyte™ System Troubleshooting
1-5
easyCyte™ Instrument
The easyCyte™ System was designed for easy operation and minimal maintenance. The
power switch, sample loader, and waste and cleaning solution vials are the only instrument
components that you will routinely handle, and most of your interaction is through the
software via the laptop. The power switch is located on the back-right side of the
instrument.
access to flow cell
power switch
(located on back of
instrument)
indicator lights
sample loader
waste vial
cleaning solution vial
guava easyCyte™ System and components
Sample Loader
The sample loader holds up to two sample tubes. The loader arm can be lowered and
swiveled, allowing you to quickly switch from one sample to the next during acquisition.
To load samples, pinch the release lever with your thumb and forefinger and gently lower
the arm assembly (figure 1). Once lowered, rotate the assembly to change the sample
tube (figure 2), then push up on the assembly until you hear a click.
release lever
1. Pinch release lever and gently lower sample holder.
1-6
guava easyCyte™ System User Guide
2. Rotate loader assembly to change tubes.
Cleaning Solution and Waste Vials
The cleaning solution vial (located on the right ) can be filled with Guava® Instrument
Cleaning Fluid (ICF), for easy system cleaning. For information on filling the vial, refer to
“Filling the Cleaning Solution Vial” on page 3-4.
The waste vial (located on the left ) captures the sample fluid after it exits the fluid
system. Empty the waste vial at the end of each day or more often, if necessary. Add
5 mL of bleach to the vial after you empty it (see page 3-5 for information).
Fluid System
Sample uptake occurs through a capillary and is
regulated by a variable-speed fluid pump. The pump does
not require sheath fluid or other supplementary fluids for
operation.
Because the system’s sampling precision depends on the
integrity of the fluid pathway, it is important to maintain a
clean system. Do not allow samples to remain in the
capillary for extended periods of time. Perform frequent
cleaning cycles to prevent the build-up of cellular debris
that may restrict sample flow. Always keep a tube on
water loaded when the instrument is not in use. If a clog
does occur, you can clear it by using the backflush
feature, which reverses the flow of fluid and flushes it out
of the flow cell at a high speed. Refer to “Backflushing the
Fluid System” on page 3-4 for detailed instructions on
using the backflush feature.
capillary
Some assays allow you to select the sample flow rate for acquisition. For most assays,
the Medium flow rate is the default. This works well when the sample concentration is
approximately 500 particles/µL. If the sample concentration is higher, dilute the sample or
use a lower flow rate. For assays where the peak CV is critical, such as Cell Cycle, use
the Low or Very Low flow rate. Always use the same flow rate for acquisition that you use
for adjusting the settings.
Laser
The guava easyCyte™ has three lasers—blue, violet, and red. During acquisition using
InCyte™ or easyCheck™, all three lasers turn on. During acquisition using ExpressPro,
the blue and red lasers turn on. During acquisition using any of the other guava® software
modules, only the blue laser turns on. The lasers turn off when acquisition is complete.
■ NOTE: For information on the easyCyte™ lasers and the fluorochromes compatible
with each, refer to "easyCyte Lasers and Fluorescent Filters" in Specifications.
Software CD
A CD contains guavaSoft™ software. The guava easyCyte™ System User Guide is
installed with the software.
easyCyte™ Instrument
1-7
guavaSoft™ Software Overview
Use guavaSoft™ software for the acquisition and analysis of data.
Main Menu
The guavaSoft™ software main menu allows you to select an assay from either a list of
favorites or the program search list. Essential Tools allows you to run easyCheck™, clean
the instrument, or select Setup, where you can customize your list of favorites. Use
links @ Millipore to quickly access EMD Millipore websites for information.
guavaSoft™ software main menu
Favorites
Favorites allows you to quickly select an assay. You can
have up to seven assays at a time in your Favorites list. To
add a favorite assay, select the assay from the Program
Search list and click Add to Favorites. The new assay will
be added to the bottom of the list. And if the list already
consists of seven assays, the first assay in the list will be
removed.
To remove a favorite assay, click Setup under Essential Tools. Remove the check from
the check box for the assay you wish to remove. Click DONE.
1-8
guava easyCyte™ System User Guide
Program Search List
The Program Search List allows you to select an assay to run or open a FlowCellect®
template in InCyte™. You can create a Favorites list to quickly select an assay later. The
main menu allows you to select from the following assays:
• guava ViaCount® Assay for performing cell counting and viability assays
• guava® ExpressPro Assay for performing assays with up to six colors, or where time,
area, and/or width parameters are necessary
• guava InCyte™ for performing assays with up to ten colors and two scatter
parameters, or where time, area, and/or width parameters are necessary, or if you
want to analyze any guava FCS 3.0 data files
• guava Nexin®, guava® Caspase, guava® TUNEL, and guava® MitoPotential Assays
for performing apoptosis assays
• guava® Cell Cycle Assay for performing DNA cell cycle assays
• guava® CellPaint Assay for tracking target cells in mixed populations, or for
monoclonal antibody (mAb) screening in mixed cultures
• guava® CellToxicity Assay for cell-mediated cytotoxicity determinations
• guava® CellGrowth Assay for cell proliferation studies and to assess the number of
live versus dead cell
Additionally, FlowCellect® template are available for acquisition and/or analysis using
InCyte™. See “FlowCellect® Templates” on page 1-11.
Essential Tools
guava easyCheck™ allows you to check the system’s counting, fluorescence, and
scatter performance prior to running samples. The guava easyCheck™ Bead is a
standard particle used with this tool.
Cleaning allows you to run Guava Clean, an automated system cleaning cycle that
cleans the fluid system.
Setup allows administrators to:
• configure certain software features for specific users (see “Appendix A: Administrator
Features” for more information)
• remove assays from your Favorites list
• enter assay registrations codes (see “Unlocking guava InCyte™” on page 1-10)
■ NOTE: The Setup button is available only when an administrator is logged onto the
system.
links @ Millipore
This section allows you to access commonly used EMD Millipore websites. You must
have internet access.
guavaSoft™ Software Overview
1-9
Check for Template Updates
Select Check for Template updates to search online or, if you are not connected to the
internet, any available USB drives, for the latest FlowCellect® templates. The system will
notify you if you currently have the latest templates installed.
1
If updated templates are available, the following dialog appears. Click Yes to install
the latest template package.
Click Export to download the templates to a USB drive.
2
Click Close when you receive confirmation that the latest templates were installed.
If you chose to export the templates to a USB drive, select the drive from the dropdown menu and select Export.
Exit
Closes guavaSoft™ software
Unlocking guava InCyte™
guava InCyte™ requires an unlock key. If you get a message containing your computer
code and prompting for an unlock key, e-mail EMD Millipore Technical Support and they
will provide you with an unlock key for InCyte™.
1 - 10
guava easyCyte™ System User Guide
FlowCellect® Templates
Software templates are available for all FlowCellect® assays. The templates can be used
for acquisition and/or analysis. Open a template before acquiring your samples in
InCyte™. The instrument settings, plots, gates, and markers for the assay are contained
in the template. If you already acquired data using either InCyte™ or another guavaSoft™
module, open the data file in the template and perform analysis using the Analysis
Method provided.
Templates for the following assays are available:
FlowCellect® Cytochrome c for mitochondrial cytochrome c loss
FlowCellect® Human CD4/CD8 T Cell for quantification on CD4 and CD8 T cells
FlowCellect® Human T Cell Caspase for caspase activation in CD4 and CD8 T cells
FlowCellect® MitoCaspase for caspase and mitochondrial membrane depolarization
detection
• FlowCellect® MitoDamage for apoptosis, mitochondrial membrane depolarization,
and cellular death detection
• FlowCellect® MitoPotential Red for mitochondrial membrane depolarization and
cellular death detection
• FlowCellect® Th1/Th17 for Th1 and Th17 intracellular cytokine detection
•
•
•
•
Using a FlowCellect® Template
For more information on using the templates, refer to the user guide for the individual
assay kit.
1
To open a template, select it from the Program Search list and click Launch.
The assay template opens in InCyte™.
2
Select the Analyse or Acquire button at the top of the control panel. Perform
acquisition or analysis according to the instructions outlined in the kit user guide.
• If you are acquiring samples, the instrument settings should be close to what you
need. Check the settings and fine tune, if necessary.
• If you are analyzing InCyte-acquired data, but you acquired the data without the
use of a template, you will need to drag the template Analysis Method to the
Analysed Group. Check the gates and markers and fine tune, if necessary.
• If you are analyzing a data file acquired using a program other than InCyte™, open
the data file. The assay template will contain the appropriate Analysis Method. You
will need to create an Analysed Group and drag the data file and the Analysis
guavaSoft™ Software Overview
1 - 11
Method to the Analysed Group. Check the gates and markers and fine tune, if
necessary.
guava InCyte™ Overview
About guava InCyte™
guava InCyte™ was developed to be an open assay module providing all the tools for
instrument setup, sample acquisition, and data analysis.
The guava InCyte™ module allows you to acquire and analyze up to ten fluorescence
parameters in combination with forward scatter (FSC) and side scatter (SSC), as well as
area, width, and time. It provides automated compensation and an instant update feature,
making it easier to perform complex analysis.
Key Features
A single Method (or analysis strategy) can be applied to multiple data sets in parallel, or
multiple Methods can be applied to a single data set to obtain statistics to be displayed in
the heat map. The process of creating equation-based gating schemes has been
simplified through use of drag-and-drop regions; “draggable” features are used
throughout InCyte™.
The HeatMap allows you to visually compare results using a plate map that displays
varying shades of blue to represent relative statistical values. Results from up to six
parameters or six data sets can be displayed simultaneously.
1 - 12
guava easyCyte™ System User Guide
Additionally, all channels can be used with reagents that fluoresce in appropriate
channels. Refer to "easyCyte Lasers and Fluorescent Filters" in the Specifications section
for filter information.
Components of InCyte™ Files
Data files acquired using InCyte™ contain raw FCS data, the analysis components also
known as Methods (regions, gates, markers, statistics, etc), instrument settings, and
compensation settings. The raw data combined with the Analysis Method make up the
InCyte™ Analysed Group.
At the start of acquisition, you have the option to select a Method, instrument settings,
and compensation settings. These components can be retrieve from a saved InCyte™
FCS file. You can also retrieve the Method and/or instrument settings from individual
saved Method and instrument settings files. If you do not retrieve a Method, a default
Method will be applied.
Tool Bar
The tool bar appearing on the far left side of the window provides additional features for
acquisition and analysis. The features are also accessible from the Tools menu. Other
than the Pie Legend, which is described on page 4-5, the tools are described in detail at
guava InCyte™ Overview
1 - 13
the point in the workflow where they are used.
Event Log
Show/Hide Acquisition Controls
Region List
Gate List
Show Current Run Stats
Show Group Stats
Show Instrument Settings
Show Compensation Controls
Show Pie Legend
Show Sample Info
Show Gain Controls
Show Miscellaneous Controls
InCyte™ Acquisition Screen
◆ WARNING: Do not run Excel® software, Internet Explorer® browser, or any other
program on the laptop while using guavaSoft™ software to acquire data from the
guava easyCyte™ System. guavaSoft™ software requires the full resources of your
laptop during data acquisition. Running other programs (even if you are not actively
using them) during a run may interfere with acquisition or interrupt the run.
The Acquisition screen appears when you enter the assay. Use the Acquisition screen to
acquire data from samples you run on the easyCyte™ System. You can also perform data
analysis from the Acquisition screen immediately following acquisition.
Menu bar
Switch between
Acquire and Analyse
control panels
Button panel
Tool bar
Control panel - displays
real-time acquisition
parameters
Plate map
Plot tool bar
1 - 14
guava easyCyte™ System User Guide
Acquisition Buttons
Place the cursor over the icon to display text describing the button.
Start New Session Adjust Settings
Retrieve
Settings
Save
Settings
Acquire Next
Sample
Capillary
Cleaning Tools
Save and Close
Current Sample
Stop and Close
Session File
Next Step
Abort
The following table describes the acquisition buttons.
Acquisition Button
Description
Start New Session File
Allows you to select the worklist file, the Method, instrument
settings, and compensation, prompts you to load the for
acquisition or the adjust settings, if applicable.
Adjust Settings
Allows you to adjust instrument settings using the appropriate
sample.
Acquire Next Sample
Begins data acquisition for the next sample. You can make
changes to the sample information (except for Events to Acquire)
after acquisition and before clicking Save and Close Current
Sample. After clicking Save and Close Current Sample, the
Sample # advances and any changes made to the sample
information will apply to the next sample.
■ NOTE: When Autostart Acquisition is checked, data
acquisition starts automatically each time a new tube is
loaded. Therefore, you do not need to click Acquire Next
Sample or Save and Close Current Sample.
Save and Close Current Saves the sample data. You can now acquire the next sample.
Sample
Next Step
Proceeds to the next step in the data acquisition process. The data
already acquired is saved.
Retrieve Settings
Allows you to recall instrument and analysis settings from a settings
file and download the settings to the easyCyte™ instrument. If this
button is disabled, your system administrator may have chosen to
not allow the retrieval of Methods and instrument settings from
individual Method and instrument settings files. These settings can
be retrieved only from an FCS file.
guava InCyte™ Overview
1 - 15
Acquisition Button
Description
Save Settings
Allows you to save the current instrument settings to a separate
file.
Capillary Cleaning
Tools
Backflush reverses the flow of fluid out of the flow cell. Perform a
backflush if the acquisition rate declines and you suspect a clog.
During acquisition, click Save and Close Current Sample before
Backflush. Follow a backflush with a Quick Clean.
Quick Clean cleans the fluid pathway. During acquisition, click
Save and Close Current Sample before Quick Clean.
Stop and Close Session Stops the assay after finishing the acquisition for the current
File
sample. After stopping you cannot resume.
Abort
Stops the session. The data for the current sample is not saved.
You cannot resume an assay after you abort. You must start the
assay from the beginning.
Autostart Acquisition
An automated sample acquisition feature saves you from having to click Acquire Sample
and Save and Close Current Sample. Check the Autostart Acquisition box to
automatically start data acquisition each time you load a tube. Be aware that if you use
the Autostart Acquisition feature, you must enter a Sample ID prior to loading the sample.
■ NOTE: If you are using Autostart Acquisition, it is automatically disabled when you
click Settings, then Adjust Settings or Retrieve Settings, Quick Clean, or
Backflush. If you click one of these buttons, you must recheck the Autostart
Acquisition box to turn on this feature again.
1 - 16
guava easyCyte™ System User Guide
Acquire Control Panel
Sample Info
AutoSave FCS 2.0 Files saves files in FCS 2.0 format, in
addition to the FCS 3.0 files that are saved.
Autostart Acquisition automatically starts acquisition when
you load each tube. You do not need to click the Acquire Next
Sample button.
Sample Info
• Sample ID: Displays the Sample ID for the individual
sample. Defaults to the plate well number.
• Sample No: Displays the sample #. This number defaults
to 1 and advances at the completion of sample acquisition.
• Total count: Displays the number of events to acquire.
The default is 5000. Total count changes to Gated count if
you apply a count gate.
• Count Gate allows you to select a gate used as a counting
gate. All events above the threshold are saved to the file
whether they are in the gate or not.
• % Acquired: A progress bar provides an estimate of the
target event count during acquisition.
• Conc.: Displays the cells/µL that have exceeded the
threshold.
• Flow Rate: Displays the flow rate selected during the
adjust settings step (µL/s).
• Volume: Displays the sample volume that is acquired.
• Dilution Factor: Displays the dilution factor. The default is
1.
• Original Volume: Displays the original sample volume.
The default is 10.
• Clear Events: Click to clear the display and restart
acquisition during adjust settings.
guava InCyte™ Overview
1 - 17
InCyte™ Analysis Screen
The Analysis screen allows you to analyze data from samples that were previously
acquired. When you open a data set, the data for the first sample appears. The samples
within the file are listed in the Data pane. Click the + in front of the FCS file to display the
list of samples. Click any sample to view the data for that sample. You can also click the
up/down arrows on the keyboard to select samples.
You can access the Analyse screen by clicking Analyse at the top of the control panel. If
you display the Analyse screen at the completion of acquisition, the samples you just ran
are listed in the Data pane. Any gates and marker you set during acquisition will appear.
Tool bar
Analysis control panel:
• Data pane
• Analysis Methods pane
• Analysed Data pane
Plate map
Plots
The plots area of the window displays three plots by default. Use the
Plots menu to change the number of plots displayed. Use the tool
bar on the right to change the plot type and set regions and gates.
For more information on plots, see “Plots” on page 4-2.
Plate Map
Undock
Plot type
New Region
Plot gate
New Stat Marker
Edit Overlay List
Zoom
The plate map provides a visual representation of the samples
contained in the dataset. During analysis, click a well in the plate
map to view the data for that sample in the plots. Place your cursor over a well to display
the results for that sample. If you are using the HeatMap feature, the plate map provides
a visual representation of the relative values for each well, or well-to-well variations in
varying shades of blue. For more information on HeatMapping, see “HeatMap” on
page 4-38.
1 - 18
guava easyCyte™ System User Guide
Analyse Control Panel
Analysis Buttons
Place the cursor over the icon to display text describing the
button. The buttons have the same function for each pane
but apply specifically to that pane.
New
Open
Save
Duplicate
Delete
Data
Displays the open data files, as well as any user-created
subsets of these files or groups and allows you to select a
data set or group for analysis.
Analysis Methods
Displays the Analysis Methods for the current experiment.
Each Analysis Method contains a gate list, a region list, and a
metric (statistical parameter). InCyte™-acquired files contain
Methods. For data files acquired using a program other than
InCyte™, you must create a new Method or open an existing
Method before you can perform analysis.
Analyzed Data
Displays the FCS file and the associated Method. Created by
pairing a non-InCyte™–acquired data file with a new or
existing Method during analysis, or created automatically
during acquisition using InCyte™.
Exiting the Assay
Before quitting the software, be sure to save your current analysis using the Save
Analysed Group icon in the Analysed Data pane.
1
To quit InCyte™ and return to the guavaSoft main menu, select Quit from the File
menu.
2
Select Yes in the confirmation dialog.
guava InCyte™ Overview
1 - 19
Files
guava InCyte™ automatically saves a flow cytometry standard (FCS) 3.0 data file, which
contains the data for all samples within a data set.
Additionally, you can optionally choose to save (or export) the following files:
• a separate Method file
• a separate instrument settings file
• FCS 2.0 or 3.0 files for individual samples for analysis using third-party analysis
programs
• list-mode data files
For information on exporting FCS 2.0, 3,0, and list-mode files, see “Exporting Results” on
page 4-33.
■ NOTE: To keep your computer performing optimally, periodically clear old files from
your hard drive by archiving the files to a back-up storage location.
Flow Cytometry Standard (FCS) 3.0 Data Files
InCyte™ saves a single FCS file containing all the samples within a data set. The
extension .fcs is automatically appended to the file name.
FCS files are data files saved in a format compatible with standard flow cytometry
analysis applications as defined by the Society for Analytical Cytology [Cytometry.
1990:11(3);323–332]. One FCS 3.0 file is saved for all samples acquired within a data set.
■ NOTE: Always save guavaSoft™ software’s data files directly to the laptop’s hard
drive during acquisition. Saving acquisition data to an outside network may result in
data loss. You may copy the file(s) to another location when acquisition is complete.
Method Files
A Method contain all the analysis components (gates, regions, markers, plots, parameters,
and statistical setup). When you acquire data using InCyte™, Methods are part of the FCS
file. Methods can also be saved to a separate file (.gsy). Data files acquired using a
program other than InCyte™ will not have Methods associated with them.
Instrument Settings Files
Instrument settings are automatically saved with the FCS file. You can also save
instrument settings to a separate file. The extension .GST is automatically appended to
the file name you enter. For more information on instrument settings, see “Saving
Instrument Settings” on page 1-23.
1 - 20
guava easyCyte™ System User Guide
Appending and Overwriting Existing Files
If you select the name of an existing dataset name at the start of acquisition, you will be
prompted to either append or overwrite it. If you append, the sample number defaults to the
next available number in the existing data file.
If you append data to an existing file, the instrument settings and analysis gates and
markers are automatically updated to reflect the settings for the last sample in the file.
■ NOTE: Your system administrator may have configured guavaSoft™ software to
disable appending and/or overwriting files. If appending only is disabled, you may
create a new file or overwrite an existing file. If overwriting only is disabled, you may
create a new file or append to a copy of an existing file. If both appending and
overwriting are disabled, you must create a new file.
Event Log
Each time you run an assay, guavaSoft™ software saves a log containing a list of all
events that occurred during the assay. This information is contained within the FCS data
file. To view this list, click the Event Log icon in the tool bar, or select Tools > Show
Event Log. A list of all events appears with the date and time the event occurred.
Allows you to filter the list.
guava InCyte™ Overview
1 - 21
You can filter the list to view errors, warnings, statuses, and/or actions. Click the
appropriate check box(es) to display the types of events you wish to view.
Every step the instrument performs, independent of the operator (for example, priming,
setting thresholds, performing calculations) is logged. Every step the operator performs
(for example, key presses, selections, changes to gates and markers, logging comments)
is also logged. During data analysis, although changes to settings are logged, the specific
details of the change may not be.
If errors or warnings occur during a run, a message appears in red in the status bar
indicating that errors/warnings have been logged and how many times they have
occurred.
Warnings include:
• Less than 10 particles/µL. Sample is too dilute. Accuracy may be compromised.
• More than 500 particles/µL. Sample is too concentrated. Please dilute or accuracy may
be compromised.
• The run timed out before enough events were acquired.
• Adjust Settings timed out. Please re-enter Adjust Settings if necessary to complete the
instrument set-up.
• Maximum velocity exceeded for “x” events (applies only to area/width parameters)
Errors include:
• The pump has reached the end of its stroke. You should run this sample again.
• Error occurred while trying to generate FCS 2.0 file for sample x.
To export the log to a text file, click Export. Select the storage location and enter a file
name. Then, click Save.
Instrument Settings
InCyte™ saves the instrument settings for each sample acquired. To access the settings
window select the Analysed Group from the Analysed Data pane, then click the Show
Instrument Settings tool in the tool bar, or select Tools > Show Instrument Settings. In
addition to the threshold, gains, and compensation, all of the acquisition information, such
as date, time, number of events, total volume, concentration, and flow rate, along with
sample-specific information, like sample ID and number, dilution factor, and original
volume are saved.
To copy the window to the clipboard, right-click in the window and select Copy to
Clipboard.
To export the settings to a CSV file for use in a spreadsheet program, click Export to
CSV. Select the folder where you want to store the file, enter a file name, and click Save.
1 - 22
guava easyCyte™ System User Guide
To print the settings, click Print Stats.
Saving Instrument Settings
Although the instrument settings are automatically saved with the FCS file, guavaSoft™
software allows you to save the current instrument settings and analysis gates and
markers to a separate file. You must perform the adjust setting step before you can save
a settings file. You can recall this file later to:
• download the instrument settings to the guava easyCyte™ System for acquisition
• apply the gates and markers to data during acquisition
1
Click the Save settings button from the Acquisition screen.
2
Enter a name for the file, select the directory where you wish to save it, and click
Save.
Retrieving Instrument Settings
1
Click the Retrieve settings button from the Acquisition screen.
2
Locate and select the file and click Open.
The settings are downloaded to the guava easyCyte™ System.
■ NOTE: If you retrieve instrument settings after you perform the adjust settings step,
you will be prompted to repeat the adjust settings step. You can choose to repeat the
adjust settings if you wish.
guava InCyte™ Overview
1 - 23
1 - 24
guava easyCyte™ System User Guide
CHAPTER 2
Getting Started
System Startup and Shutdown
Startup
1
Turn on the power conditioner if it is not already on. Once it is powered on, it can
remain on.
2
Turn on the laptop computer.
3
Turn on the guava easyCyte™ System.
The power switch is located on the back of the
instrument at the lower-right corner (as shown).
4
When the indicator lights turn on, start
guavaSoft™ software by double-clicking the
guavaSoft 3.1 application icon on the desktop.
You can also click the Start button, point to
Programs, then Millipore, then guavaSoft 3.1,
then click guavaSoft.
power
switch
Ensure the cleaning vial is filled with ICF and
the waste vial is empty, except for 5 mL of
bleach.
■ NOTE: If the software detects a communication problem with the easyCyte™ System
or that the system is not turned on, the following message appears.
5
guavaSoft™ will start but you will only be able to access an assay’s analysis mode. If
you wish to perform acquisition, exit guavaSoft™ software then restart it. If the
System Startup and Shutdown
2-1
message appears again, shut down the system. Ensure that the USB cable between
the computer and the instrument is securely connected before restarting the
computer. When the computer is finished booting up, turn on the guava easyCyte™
System. When the indicator lights are on, start guavaSoft Software.
Shutdown
Clean the easyCyte™ System and leave a tube of water in the sample loader.
1
Run the cleaning procedure at the end of the day. See page 3-2 for complete
instructions.
After the cleaning procedure is complete, return to the Main Menu and exit
guavaSoft™. Then turn off the instrument and shut down the laptop, in any order.
2
Leave the tube of deionized (DI) water loaded on the easyCyte™ System.
The system can be left in this state.
◆ WARNING: Do not leave Guava® ICF, bleach, or any other cleaning agent on the
easyCyte™ System overnight or for an extended period of time. Prolonged exposure
to strong oxidizing agents will damage the flow cell. Always leave a tube of DI water
on the easyCyte™ System when shutting down the instrument. Change the tube of
water regularly to ensure it is clean and free of particles.
3
Exit guavaSoft™ software. Do not shut down the guava easyCyte™ System while
guavaSoft™ software is running.
Quitting guavaSoft™ Software
2-2
1
Click Main Menu from any of the guava® assay screens, the Guava Clean screen, or
the easyCheck screen.
2
Click EXIT in the lower-left corner of the main menu.
guava easyCyte™ System User Guide
Running the easyCheck™ Procedure
Run the easyCheck™ procedure at the start of each day you use the guava easyCyte™
System to ensure the system is performing properly. easyCheck™ averages the results
from three acquisitions of a guava easyCheck™ Bead sample to determine if the results
are within the expected range.
■ NOTE: Before running the easyCheck™ Procedure, perform a Quick Clean using
distilled water to prime the fluid system. If it has been more than a day since you used
the system, perform two Quick Cleans using water to prime the fluid system.
1
Prepare a 1:20 dilution of guava easyCheck™ Beads. Refer to the easyCheck™ Kit
package insert for information.
2
Click easyCheck from the main menu.
Enter bead lot #,
expiration date, and
particles/mL.
The first time you run the easyCheck™ Procedure, enter the easyCheck™ Bead Lot
# and Bead Expiration Date (found on the easyCheck™ Beads Reagent vial label),
and
Expected Particles/mL in the appropriate fields. Thereafter, enter any necessary
changes to these values.
Optionally, you may enter the guava easyCheck™ Kit lot number and expiration date
(found on the side of the guava easyCheck™ Kit box).
The Expected Particles/mL is typically around 50,000, however check the information
card that comes with the easyCheck™ Kit for the actual particle count for each new
lot. The particles/mL corresponds to the concentration of beads in your prepared
sample where the guava easyCheck™ Bead Reagent was diluted 1:20 with Guava®
Check Diluent.
■ NOTE: Your system administrator may have configured guavaSoft™ software to
require that you enter values in these fields each time you run the easyCheck™
Procedure. If the fields are blank when you access the easyCheck screen, you must
enter the current information.
3
Running the easyCheck™ Procedure
2-3
4
Mix the easyCheck™ bead sample using a vortex mixer and load the sample on the
easyCyte™ System. Click Run 1st Replicate.
The system acquires 1000 events and displays the results in the row for Replicate 1.
Check the Particle Count in the Sample Information control panel. The FSC, SSC,
BLU-V, GRN-V, YEL-V, RED-V, GRN-B, YEL-B, RED-B, NIR-B, RED-R, and NIR-R
counts should be within 100 particles of each other when acquisition is complete.
5
Remove the sample, mix it well using a vortex mixer to resuspend the beads, and
load it on the easyCyte™ System. Click Run 2nd Replicate.
The system acquires 1000 events and displays the results in the row for Replicate 2.
6
Remove the sample, mix it again to resuspend the beads, and load it on the
easyCyte™ System. Click Run 3rd Replicate.
The system acquires 1000 events and displays the results in the row for Replicate 3.
The % CV, average Particles/mL, and average FSC, optional SSC, and fluorescence
intensities for the three replicates are displayed.
■ ACQUISITION NOTES
• The progress bar provides an estimate of the target event count during the
acquisition period, which times out after 1 minute.
• If the acquisition rate appears to slow dramatically or there are no particles being
acquired, the fluid pathway may be blocked. Click Abort, load a tube of deionized
(DI) water, then click Backflush. When the backflush is complete, load a tube of
fresh DI water and click Quick Clean. Then, reload the sample and click Adjust
Settings to start the acquisition process from the beginning.
easyCheck™ Results
The software displays the %CVs and the averages for the particles/mL (bead count), FSC
and SSC intensities, and all mean fluorescence intensities (MFIs) for the three replicates.
• If any result for Particles/mL falls outside ±10% of the expected value, the result is
outside the acceptable range and appears in red. For example, if the actual particle
count is 50,000, the acceptable Particles/mL range (±10%) is 45,000 to 55,000.
• If any result for MFI falls outside ±15% of the expected value, the result is outside the
acceptable range.
• If the %CV for Particles/mL is >10%, it appears in red.
• The %CV for FSC and SSC Intensities and MFIs for the three replicates should be
<5%.
2-4
guava easyCyte™ System User Guide
Refer to the information card that comes with the guava easyCheck™ Kit for the
acceptable intensity ranges for each parameter. This information may change from lot to
lot.
To monitor the instrument performance, look at the average and %CV values for FSC and
SSC Intensity, and BLU-V, GRN-V, YEL-V, RED-V, GRN-B, YEL-B, RED-B, NIR-B,
RED-R, and NIR-R MFIs. Refer to “Viewing and Exporting easyCheck™ Results” on
page 2-5 for information on displaying past easyCheck™ results.
If the Particles/mL (count) for a replicate or the average falls outside the acceptance range,
or if an intensity value is outside the acceptable range, run Quick Clean (refer to page 3-1).
Rerun the easyCheck™ Procedure after cleaning is complete. If values continue to fall
outside the acceptance range, refer to “easyCheck™ Procedure Troubleshooting” on
page 2-8, for more information.
If the signal intensity for any of these parameters shows significant drift over time beyond
the range listed, and this change is not correlated to a change in the bead lot, a new flow
cell, or instrument service, contact EMD Millipore.
Viewing and Exporting easyCheck™ Results
To display a history of all easyCheck™ runs and view the results for individual runs, click
Show History at the bottom of the easyCheck screen. The History List control panel
opens showing a list of all easyCheck™ Procedure runs. To display the results for a
particular run, click on the run in the list.
Click to display
the results for a
particular run.
Running the easyCheck™ Procedure
2-5
• Click Log Comment to enter comments related to the run and save these comments
to the event log.
• Click View Event Log to display the event log, which lists all errors, warnings,
statuses, and actions that occurred during the easyCheck™ run. For more information
on the event log, refer to “Event Log” on page 1-21.
• Click Show Trend Graph to display a trend graph of the Particles/mL value from the
last 30, 60, or 90 runs. See “Viewing Trend Data” on page 2-7.
• Click Print Screen to print the results. A print dialog box appears allowing you to
select the printer.
• Click Export to Spreadsheet to export the data from all easyCheck™ runs to a
spreadsheet file. The file contains the average and %CV for each parameter, as well
as the details for each replicate of all runs.
2-6
guava easyCyte™ System User Guide
• Click Export Service Check File to export the Service Check file. The Service Check
file is a zipped file containing the detailed results from the most recent easyCheck™
run. This file is used by service personnel to troubleshoot your system. If your
easyCheck™ results continue to fail. Use this export feature to export the file so that
you can send it to EMD Millipore.
Viewing Trend Data
To view a trend graph of the Particle/mL data, click Show History at the bottom of the
screen, then click Show Trend Data.
A trend graph appears showing the Particles/mL results from the last 30, 60, or 90 runs. A
data point appears for each of the three replicate values. The date appears for every
seventh or eighth time that the easyCheck™ Procedure was run.
Legend
Display data from
last 30, 60, or 90
runs.
A legend in the lower-right corner of the window lists the information found on the graph.
A description of the items in the legend appears in the following table.
Legend Item
Description
Replicates Outside Range
data point appears as a red triangle (value falls outside the
high or low, 10% limit lines)
Replicates Within Range
data point appears as a black triangle
Median of Replicates
a black line connects the median values from each triplicate
Limit High
pink line that appears 10% above expected particle count
Target
green line at the expected particle count entered
Limit Low
pink line that appears 10% below expected particle count
Running the easyCheck™ Procedure
2-7
easyCheck™ Procedure Troubleshooting
Problem
Possible Cause
No event counts appear for 1. Wrong beads used.
RED-R and NIR-R.
2. Red laser not operating or
problem with the signal.
One or more Particles/mL
1. System is not clean.
results falls outside the
acceptance range (appears
in red).
2. Incorrect information
entered in easyCheck™
fields.
3. Bead suspension
incorrectly prepared.
1. Use easyCheck™ beads.
Do not use Guava® Check
beads.
2. Contact EMD Millipore
Technical Support.
1. Run Quick Clean. If results
are still outside range, run
Clean Only.
2. Ensure correct Bead Lot #
and Expected Particles/mL
are entered. Refer to
page 2-3 for information.
3. Prepare fresh bead sample
and rerun easyCheck™
Procedure. Refer to
easyCheck™ Kit package
insert for preparation
instructions.
FSC, SSC, GRN-B, YEL-B, 1. System is not clean.
RED-B, NIR-B, RED-R,
and/or NIR-R intensity is
>10% outside the
2. Problem with detector or
acceptable range.
laser.
1. Run Quick Clean. If results
are still outside range, run
Guava® Clean.
2. If problem persists, contact
EMD Millipore Technical
Support.
Particle counts for FSC,
SSC, GRN-B, YEL-B,
RED-B, NIR-B, RED-R,
and/or NIR-R intensity is
not within 100 events of
each other.
1. Remove metal plate.
Unscrew tubing from top of
flow cell and firmly push
down on top of flow cell
assembly.
If problem persists, contact
EMD Millipore Technical
Support.
2. Rerun easyCheck™
Procedure. If counts are
still low, contact EMD
Millipore Technical
Support.
1. If FSC count is low,
capillary may not be
seated correctly.
2. If any of the counts is low,
possible problem with
detector.
2-8
Solutions
guava easyCyte™ System User Guide
Problem
Possible Cause
Solutions
Few events, as indicated in 1. Clogged flow cell.
1. Remove sample, load
Particle Count section of
bleach, click Backflush.
Sample Information control
Follow with Quick Clean
panel.
using DI water.
2. Insufficient sample
2. Minimum sample volume is
volume.
90 µL.
3. Beads in suspension have 3. Click Abort. Remove
settled.
sample, mix, reload, and
reacquire.
No events, as indicated in
Particle Count section of
Sample Information control
panel.
1. Ensure tube is loaded and
loader assembly is up.
2. Minimum sample volume
is 90 µL.
3. Ensure correct sample is
loaded.
4. Remove sample, load
4. Clogged flow cell.
bleach, click Backflush.
Follow with Quick Clean
using DI water.
5. Remove flow cell and
5. Broken flow cell.
inspect for damage.
Replace, if necessary.
6. Run Quick Clean and
6. Sample pump not
watch for fluid in waste
working.
vial.
7. Contact EMD Millipore
7. Laser is not operational.
Technical Support.
8. Loose fitting on minstac
8. Ensure tubing connector is
tubing (under metal plate).
secure.
1. Sample tube not properly
loaded.
2. Insufficient sample
volume.
3. No beads in sample.
Message:
The user was not assigned
Contact your system
The login user does NOT
access control to the system. administrator for user access
have read/write permission
to the software.
to the file
GuavaCheckLog.csv in the
Log folder. Contact the
system administrator for
assistance.
easyCheck™ Procedure Troubleshooting
2-9
2 - 10
guava easyCyte™ System User Guide
CHAPTER 3
Cleaning and Maintenance
Depending on the type of cleaning you wish to do, guavaSoft™ software offers different
cleaning options—Quick Clean, which you can use as often as you like throughout an
assay or anytime, and Guava® Clean, a more thorough cleaning, which you run at the
start and end of each day to thoroughly clean the fluid system.
To clean the outside of the instrument, wipe it down with a soft cloth moistened with 70%
alcohol. Follow with a cloth moistened with water.
◆ WARNING: To avoid exposure to laser radiation or electric shock, DO NOT open the
easyCyte™ System or attempt to perform any internal maintenance. There are no
user serviceable parts.
Running Quick Clean
Quick Clean is a short cleaning cycle that allows you to clean the fluid system during and
after an assay, or as often as you like throughout the day. If you are running lots of
samples or particularly dirty or adherent cells, run Quick Clean frequently. The Quick
Clean feature, which is accessible from the easyCheck™ and all guavaSoft™ Assay
screens, prompts you for a tube of cleaning solution.
1
Load one of the following solutions on the easyCyte™ System:
• deionized water to quickly flush out the fluid system
• undiluted Guava® ICF to clean the system, followed by water to rinse
• a 10% bleach solution in Guava® ICF (1 part bleach in 9 parts Guava® ICF; for
example, 100 µL bleach plus 900 µL Guava® ICF) to clean and sanitize, followed
by water to rinse
2
Click Quick Clean from any of the guava® assay screens. From guava InCyte™, click
the Capillary Cleaning Tools button and select Quick Clean.
A message appears prompting you to load a tube of cleaning solution. Ensure the
tube is loaded and click OK.
The solution is pumped through the system for approximately 30 seconds.
3
If you used water, you are finished. If you used Guava® ICF, either straight or with
bleach, load a tube of water and click Quick Clean again to rinse.
You may continue running samples, or leave the tube of water on the easyCyte™ System
until you are ready to use the system again.
Running Quick Clean
3-1
Running Guava® Clean
Run Guava® Clean to clean the instrument at the beginning and end of the day, as well as
between assays if a thorough cleaning is needed. You can also use Guava® Clean to
prime the fluid system or if you suspect there is air in the fluid lines. Fill the cleaning vial
and sample tube with water and run Guava® Clean to prime. Guava® Clean takes
approximately 15 minutes to complete. While it is running, the lasers are turned off.
Always leave a tube of deionized water on the easyCyte™ System at the end of the
cleaning cycle.
3-2
1
Click Cleaning from the main menu.
The Guava Clean screen appears.
2
Click Start Cleaning.
A dialog box appears, prompting you to load a tube of water.
3
Load a 1.5-mL tube filled with DI water in the sample loader and click OK.
guava easyCyte™ System User Guide
After approximately 4 minutes, a message appears prompting you to load a tube of
cleaning solution.
4
Load a tube half-filled with a 10% bleach solution in Guava® ICF (1 part 100% bleach
in 9 parts Guava® ICF) and click OK.
After approximately 3 minutes, a message appears prompting you to load a tube of
clean water.
5
Load a tube of fresh water and click OK.
If you notice air moving through the cleaning vial tubing, when the cleaning cycle is
complete, check the connectors holding the tubing to ensure they are tight.
After approximately 5 minutes, a message appears prompting you to load the tube of
clean water.
6
If you are shutting down the instrument, place a tube of deionized (DI) water on the
easyCyte™ System. Return to the Main Menu and exit guavaSoft™. Then turn off the
instrument and shut down the laptop, in any order.
◆ WARNING: Do not leave Guava® ICF, bleach, or any other cleaning agent on the
easyCyte™ System overnight or for an extended period of time. Prolonged exposure
to strong oxidizing agents will damage the flow cell. Always leave a tube of deionized
water on the easyCyte™ System after cleaning and when shutting down the system.
Change the tube of water regularly to ensure it is clean and free of particles.
Running Guava® Clean
3-3
Backflushing the Fluid System
The backflush feature reverses the flow of fluid out of the flow cell. Click the Backflush
button, located on each assay screen and the easyCheck screen, when you suspect that
the fluid pathway is clogged or blocked.
1
If you suspect a clog, click Abort.
2
Remove the sample tube and load a tube of 20% bleach.
■ NOTE: If you are running the easyCheck™ Procedure, use water when backflushing the system.
3
Click Backflush from any of the guava® assay screens. From guava InCyte™, click
the Capillary Cleaning Tools button and select Backflush.
A message appears instructing you to load a tube of water or bleach. Ensure the tube
is loaded and click OK.
4
If you used bleach in step 2, load a tube of deionized water and click Quick Clean to
rinse any residual bleach from the capillary.
5
Throw out the tube you used in step 2, as it may contain debris from the backflush.
6
Replace the sample and click Acquire Next Sample (If you are running the
easyCheck™ Procedure, start from the adjust settings step).
Filling the Cleaning Solution Vial
Fill the cleaning solution vial with Guava® ICF when it is approximately 1/4 full. The
cleaning solution is aspirated through the tubing in the vial. Do not allow the vial to empty.
Check it frequently to ensure it does not run dry. This will create air bubbles in the fluid
system and require that you prime the system with water. One vial of cleaning solution will
allow you to perform approximately 15 cleaning cycles.
1
Gently pull up on the vial to remove it from the bracket.
Slide cleaning solution vial out of bracket.
3-4
guava easyCyte™ System User Guide
2
Unscrew the vial from the cap.
Unscrew cleaning solution vial.
3
Fill the vial with Guava® ICF to just below the bottom of the cap. Do not overfill the
vial.
4
Replace the vial on the cap assembly and install the vial on the system.
Ensure the tubing that goes into the vial is still attached to the cap.
Emptying the Waste Vial
Empty the waste collection vial at the end of each day, or as needed.
◆ WARNING: Handle all biological specimens and materials they come in contact with as
if capable of transmitting infection. Dispose of these materials using proper
precautions in accordance with federal, state, and local regulations.
1
Gently pull up on the vial to remove it from the bracket.
Slide waste vial out of bracket.
2
Unscrew the vial from the cap.
Unscrew waste vial.
Emptying the Waste Vial
3-5
■ NOTE: Fluid may seep from the cap while it is disconnected from the vial. To prevent
waste fluid from dripping on the work surface, place the cap in a small container or on
an disposable, absorbent pad.
■ NOTE: If you notice a leak on top of the waste vial cap, tighten the connector on the
cap.
3
Empty the contents according to your local and state biohazardous waste disposal
guidelines.
4
Rinse the waste vial with water.
5
Add approximately 5 mL of bleach to the waste vial, replace the vial on the cap
assembly and install the vial on the easyCyte™ System.
Cleaning the Flow Cell
About the Flow Cell
The flow cell assembly consists of the shuttle, the
capillary, and the minstac tubing. The shuttle
houses the optical window, where the laser beam
intersects the sample. When handling the flow
cell assembly, grasp it towards the top of the
shuttle, where the minstac tubing is attached, to
avoid getting fingerprints on the optics window.
When installing a flow cell, ensure this tubing
connection is tight to avoid fluid leaking from the
top of the shuttle.
A flow cell removal tool is provided and can be
used to remove the flow cell. A tightening tool is
also provided and can be used to tighten the
minstac tubing to the instrument during flow cell
installation.
minstac
tubing
shuttle
optics
window
capillary
Using the Syringe Tool to Clean the Flow Cell Assembly
Use the syringe assembly tool to clean the flow cell. When removing the flow cell, handle
it with care. The capillary tube is fragile; avoid touching it unnecessarily. Do not force the
flow cell into the receptacle. However, slight pressure may be required to properly seat
the flow cell once it has been inserted into the receptacle.
◆ WARNING: To avoid exposure to laser radiation, turn off the power to the easyCyte™
System before attempting to remove the flow cell.
3-6
1
Lower the sample loader arm.
2
Remove the metal plate from the top of the instrument.
guava easyCyte™ System User Guide
3
Remove the tubing from the clamp and disconnect it from the instrument.
4
Remove the flow cell by grasping it at the top with your fingers and pulling straight up.
Do not pull up on the tubing.
You can also use the flow cell removal tool to remove the flow cell.
• Grasp the flow cell with the removal tool by placing the clamps into the cutouts at the
top of the flow cell, as shown.
• Pull the tool straight up to remove the flow cell.
cutouts in flow cell
5
Fill the syringe cleaning tool with water or Guava® ICF. Connect the syringe to the
minstac tubing on the flow cell. Ensure the fitting is tight.
6
Use a Kimwipe® wipe to hold the flow cell at the top of the shuttle to capture fluid that
may leak. Apply gentle, steady pressure to the plunger and watch as the fluid flows
from the tip of the capillary.
• Make sure the fluid stream is straight. If it is not straight, the tip of the flow cell may
be chipped or there may be a partial clog in the flow cell.
Cleaning the Flow Cell
3-7
• Check the capillary to ensure there are no leaks along the length of it.
• Make sure fluid is not leaking where the tubing is connected at the top of the flow
cell.
7
Unscrew the syringe from the minstac tubing. Leave the minstac tubing attached to
the flow cell.
8
If you noticed leaks at the tubing connection and you
have the tool shown at the right, use the tool to
tighten the tubing connection. A tight connection
ensures that fluid will not leak from the top of the
flow cell.
• Point the tool’s threaded stem up (away from the
flow cell) and insert the tubing connector into
clamp end of the removal tool.
• Squeeze the handles of the tool to secure the flow
cell, while tightening.
• Tightening is sufficient when you feel the tool
slipping on the connector.
clamped end
of tool over
tubing
connector
If you don’t have the tool shown at the right, use
your fingers to hand-tighten the tubing connection.
9
Use a Kimwipe® wipe to dry the end of the tubing that you disconnected from the
syringe.
Dry connector before reinstalling the flow cell.
10 If the flow cell is clean and intact, reinstall it. If it is damaged or not functioning
properly, discard it and replace it with a new flow cell.
• Install the clean (or new) flow cell by correctly positioning it vertically above the
instrument and carefully lowering it into the receptacle. The flow cell fits only one way
into the receptacle. Avoid bumping the capillary as you install it.
3-8
guava easyCyte™ System User Guide
• Use your fingers to push down on the top of the flow cell on either side of the tubing
until the flow cell clicks into place. Do not press down on the tubing at the top of the
shuttle. Press down until the flow cell stops and won’t go any farther.
11 Connect the tubing to the instrument. Make sure the tubing is screwed on tightly. If
necessary, use the tightening tool. Then insert the tubing into the clamp.
12 Ensure that the cleaning solution vial is full, then run Quick Clean to prime the system.
If starting the instrument after it has been shut down, run Guava® Clean to prime.
Watch the tubing during the Quick Clean to ensure no bubbles appear in the tubing.
13 To ensure that the flow cell was correctly installed, run the easyCheck™ procedure.
While the first replicate is being acquired, watch for bubbles in the minstac tubing. If
bubbles or leaks are visible, the tubing may not be adequately tightened.
Cleaning the Flow Cell
3-9
Replacing the Flow Cell
You can replace the flow cell if it becomes damaged or clogged so severely that
backflushing and cleaning the system do not fix the problem. When replacing the new
flow cell, handle it with care. The capillary tube is fragile; avoid touching it unnecessarily.
Do not force the flow cell into the receptacle.
◆ WARNING: To avoid exposure to laser radiation, turn off the power to the easyCyte™
System before attempting to remove the flow cell.
1
Lower the sample loader arm.
2
Remove the metal plate from the top of the instrument.
3
Remove the minstac tubing from the clamp and disconnect it from the instrument.
4
Remove the flow cell by grasping it at the top with your fingers and pulling straight up.
Do not pull up on the tubing.
You can also use the flow cell removal tool to remove the flow cell.
• Grasp the flow cell.with the removal tool by placing the clamps into the cutouts at the
top of the flow cell, as shown.
• Pull the tool straight up to remove the flow cell.
cutouts in flow cell
3 - 10
guava easyCyte™ System User Guide
5
Install a new flow cell by correctly positioning it above the instrument and carefully
lowering it into the receptacle. The flow cell fits only one way into the receptacle. Keep
the flow cell completely vertical and avoid bumping the capillary against the
instrument or sides of the receptacle. Use your fingers to press down on the top of the
flow cell on either side of the tubing until the flow cell clicks into place. Do not press
down on the tubing at the top of the shuttle.
6
Connect the tubing to the instrument, then insert the tubing into the clamp.
7
Ensure that the cleaning solution vial is full, then run Quick Clean to prime the system.
If starting the instrument after it has been shut down, run Guava® Clean to prime.
8
To ensure that the flow cell was correctly installed, run the easyCheck™ procedure.
While the first replicate is being acquired, watch for bubbles in the minstac tubing. If
bubbles or leaks are visible, the tubing may not be adequately tightened.
Replacing the Flow Cell
3 - 11
Replacing the Fuses
The AC fuse is located on the rear panel to the right of the AC power cord connector.
◆ WARNING: Turn off the power switch at the back of the instrument and disconnect the
power cord.
fuse holder
power switch
AC power cord connector
1
Remove the fuse holder cover using a small screwdriver and pivot to expose the fuse
carrier.
2
Pull out the fuse carrier.
3
Carefully remove the fuses and replace with new fuses. Use the same type and rating
as listed in the Specifications section.
■ NOTE: If you have a 110 V supply you will need to replace a single fuse. If you
have a 220 V supply you will need to replace two fuses.
3 - 12
4
Insert the fuse carrier back into the fuse holder. The fuses will face the inside of the
fuse holder.
5
Replace the fuse holder cover.
6
Reconnect the power cord and turn on the main power switch.
guava easyCyte™ System User Guide
Returning the System for Service
Contact EMD Millipore Technical Support for the Decontamination Form and instructions.
The form must be returned via e-mail before you can return the instrument. A return
authorization (RMA) number will be issued to you. Write this number on the outside of the
shipping box. If you did not save the original shipping box, you can order one (see
Appendix C: Ordering Information).
1
Perform the Guava® Clean procedure (see “Running Guava® Clean” on page 3-2).
2
Clean and disinfect the cleaning solution vial and waste vial.
3
Power off the laptop and instrument.
4
Disconnect the laptop, instrument, and power conditioner.
5
Pack the laptop and power cord in the laptop box.
6
Pack the USB cable, extension cable, and power cord in the accessory box.
7
Remove the flow cell from the instrument. Use the syringe cleaning tool to flush it with
water, and then flush it with air. Store the flow cell in the capillary box. For information
on cleaning the capillary, see “Using the Syringe Tool to Clean the Flow Cell
Assembly” on page 3-6.
8
Tape the capillary hatch closed.
9
Place the instrument in the shipping bag and secure the bag. Be sure to include the
cleaning solution and waste vials.
10 Place the instrument in the shipping box. Always use two people to lift the instrument.
11 Place the blue shipping foam over instrument. Place the accessory box on top.
12 Place the cover on the box and insert the white locks into the holes.
Returning the System for Service
3 - 13
3 - 14
guava easyCyte™ System User Guide
CHAPTER 4
guava InCyte™ Assay
Introduction
guava InCyte™ was developed to be an open assay module providing all the basic tools
for sample acquisition and data analysis. The guava InCyte™ module allows you to
acquire and analyze up to ten fluorescence parameters in combination with forward
scatter (FSC) and side scatter (SSC), as well as area, width, and time.
The instrument is configured to detect the following fluorochromes or fluorochromes with
similar fluorescence. The filter is listed first, followed by the laser. For example, BlueViolet (BLU-V) is the filter for the blue fluorescence emission off the violet laser.
For a complete list of the fluorochromes compatible with each laser, see "Fluorochromes"
in the Specifications section.
Filters off the violet laser:
• Blue-Violet (BLU-V) parameter – DAPI, Brilliant Violet 421, Pacific Blue, Cascade
Blue, or Alexa Fluor® 405
• Green-Violet (GRN-V) parameter – Brilliant Violet 510 or Pacific Green
• Yellow-Violet (YEL-V) parameter – Brilliant Violet 605 or QDot 565
• Red-Violet (RED-V) parameter – Brilliant Violet 650 or eFluor 650
Filters off the blue laser:
• Green-Blue (GRN-B) parameter – FITC, GFP, or Alexa Fluor® CF-488
• Yellow-Blue (YEL-B) parameter – Phycoerythrin (PE)-based, Cy3, Alexa Fluor® 532,
propidium iodide (PI), TRITC, and DS Red reagents
• Red-Blue (RED-B) parameter – PE-Cy5, PerCP, 7-AAD, PE-Cy5.5, and PI
• NIR-Blue (NIR-B) parameter – PE-Cy7
Filters off the red laser:
• Red-Red (RED-R) parameter – Allophycocyanin (APC), Cy5, or Alexa Fluor® CF647.
• NIR-Red (NIR-R) parameter – APC-Cy7, or APC-Alexa Fluor® 750
Introduction
4-1
InCyte™ Application Window
The application window opens displaying three plots. A tool bar at the left edge of the
window allows you to access most of the application controls (see Figure 4-1). You can
find these same options in the Tools menu in the menu bar.
The control panel displays the controls for acquiring and analyzing data. Click Analyse or
Acquire to display the desired control panel. Click in the top bar (above the Analyse and
Acquire buttons) to drag the floating control panel to any area of the screen or dock it on
the right side of the window. To redock it on the left, double-click the bar at the top of the
panel. To hide the control panel, click the X in the top-right corner. To redisplay the panel,
choose Tools > Show/Hide Acquisition Controls from the menu bar, or click the
Acquisition Controls icon in the tool bar on the left edge of the application window. Place
your cursor over any button in the Analyse or Acquire button panel to see text describing
the button.
Menu bar
Switch between
Acquisition and
Analysis
Button panel
Tool bar
Displays real-time
acquisition parameters
Plate map
Plot tool bar
Figure 4-1 InCyte™ application window – Acquisition screen
Plots
Use the Plot menu in the menu bar to set up the plot layout. Select the number of plots to
display (2x2 to 5x5 represents the number of plots displayed down vs across). Up to 24
plots can be displayed at one time.
4-2
guava easyCyte™ System User Guide
Plot Tool Bar
• Use the Undock icon to detach and enlarge the plot. The plot
Undock
will detach in its own floating window. Drag the bottom-right
Plot type
corner to increase the size of the plot window. Copy the
zoomed plot to the clipboard for higher resolution plots. To
New region
reattach the plot, click the Undock icon again or the X in the
Plot gate
top-right corner.
New Stat Marker
• Use the Plot type icon to select Dot Plot, Histogram, or
Contour Plot, and to adjust settings such as smoothing
Edit overlay list
(histograms only)
histograms and contours and selecting the dots to display in
dot plots. Contour plots can be used only during analysis. See
Zoom
“Changing the Display of Data in Plots” on page 4-3 for
information.
• Use the New region and Plot gate icons to create regions and apply gates,
respectively. See “Regions, Gates, and Statistics” on page 4-19.
• New Stat Marker is described in “Stat Markers” on page 4-26. Edit overlay list is
described in “Overlaying Histograms” on page 4-49.
• To change the plot parameters once data is displayed, click the parameter name
and select the new parameter from the pop-up menu. To customize a parameter
name, see “Changing Parameter Names” on page 4-5.
• To copy a plot to the clipboard, right-click the plot and select Copy Plot to
Clipboard.
• Use the Zoom icon to zoom an area of the plot. Click and drag a rectangle in any
direction to encompass the population you wish to zoom in on. To scroll the data in
the plot, click the icon, then hold down the Shift key while you click and drag. To
unzoom, click the Zoom icon again, then press and hold the Ctrl key while you click
anywhere in the plot.
Changing the Display of Data in Plots
You can make the following changes to the display of data
in these plots. The settings are saved with the Analysed
Group. Click the Plot Type icon in the plot tool bar and
select Plot Settings.
InCyte™ Application Window
4-3
•
Set the number of dots displayed in a dot plot
Use the slider to set the number of dots from 1 to 100%. The default is 100%.
•
Smooth histograms
Use the slider to set the amount of smoothing (sigma) from 0 to 5.0, where 0 is no
smoothing. The default is 1.5.
•
Smooth contour plots
You can adjust both the number of levels from 5 to 20, and the amount of smoothing
(sigma) from 0 to 5.0, where 0 is no smoothing. The defaults are 10 and 1.5,
respectively. Contour plots can be created during analysis only.
4-4
guava easyCyte™ System User Guide
Changing Parameter Names
You can change the default long (or stain) name of any parameter. The short name, for
example, GRN-B-H, is fixed and will still appear in the plots in parentheses after the new
long name.
1
Choose Application > Change Parameter Names
from the menu bar.
The Parameter Names dialog box appears. Area and
width are listed only if they were selected during the
adjust settings step prior to acquisition.
2
Double-click the existing name in the Long or Stain
Name text field and type in the new name, or use the
tab key to highlight the desired field and begin typing
the new name.
The short name will appear in parentheses following the
long name under the plots.
3
Click Update.
Pie Legend
The Pie Legend contains the HeatMap legend and the IC-50
application. The HeatMap legend (circle) is used to display the data
results for each Analysed Group (data + Method) in the plate map.
Use the Show Pie Legend icon in the tool bar to display this
workspace.
The HeatMap legend shows one sector by default. You can create up
to six sectors for six experiments. Each sector will have a data file (or
group) paired with an Analysis Method. To divide the legend into
multiple sectors click N=2, 3, etc. To clear a sector, right-click it and
Show Pie Legend
InCyte™ Application Window
4-5
select Clear Sector. For more information on Heatmapping, see “HeatMap” on page 438.
Select the number of sectors to display.
Drag and drop a data file (or group) and an Analysis
Method to the HeatMap legend for analysis.
Click to change the display from single- to multi-sector
display. Up to six sectors can be displayed.
Set minimum and/or maximum thresholds to eliminate
data above and/or below the thresholds.
• N allows you to select the number of sectors you want to display. Sectors are
numbered (1-6) with each new sector entering the upper-left portion of the circle.
• Threshold allows you to apply upper and/or lower limits on statistical values to
eliminate data above or below the thresholds. The Current panel is only functional when
one sector is being displayed, or when multiple sectors are used but the HeatMap
legend is in single-mode display. See “Thresholds” on page 4-43 for information.
• The IC-50 application allows you to rapidly convert time course or dose response
studies into standard curves for the calculation of compound IC50/EC50 values. See
“IC-50/EC-50” on page 4-44 for more information.
guava InCyte™ Sample Acquisition
4-6
1
Open guavaSoft™ software by double-clicking the guavaSoft™ icon on the desktop.
2
Click InCyte from your list of Favorites, or select InCyte™ from the Program Search
list and click Launch.
The guava InCyte™ window opens in Acquisition mode, if the easyCyte™ System is
turned on. The top portion of the control panel contains 10 buttons used to control
acquisition, adjust, retrieve and save instrument settings, and select cleaning options.
guava easyCyte™ System User Guide
For information on the control panel, refer to “Acquire Control Panel” on page 1-17.
3
Prepare samples for acquisition in tubes.
4
Click the Start New Data Session File button.
A dialog box appears allowing you to select a data file name and location, Analysis
Method, and instrument and compensation settings.
5
An FCS data file will be named and stored automatically. The default storage location
is My Documents. The default file name is the date and time. To change the defaults,
click Select Output FCS file, enter the new information, and click Save.
 NOTE: Always save guavaSoft™ software data files directly to the system’s hard drive
during acquisition. Saving data to a network or location other than the computer’s
guava InCyte™ Sample Acquisition
4-7
hard drive may result in data loss. You may copy the file(s) to another location when
acquisition is complete.
6
Select the information you want to retrieve:
 NOTE: Your system administrator may have configured guavaSoft™ software to
disable the retrieval of Methods, instrument settings, and compensation settings
from Method files and individual instrument settings files. If the Settings and
Compensation retrieval options are disabled, you will not be able to adjust
instrument settings. However, you can retrieve settings and Methods from a single
FCS file.
• Method – either from an InCyte™ FCS file or a saved Method file (.gsy). Click
Choose, locate the file, then click Open.
The FCS or Method file must be from InCyte™, version 3.0, or later. If you do not
choose a Method file, a default one will be created automatically.
If you select an FCS file to obtain the Method, you can also retrieve the instrument
settings and compensation settings that were saved with that FCS file.
• instrument settings – either from an InCyte™ FCS file or a saved instrument
settings file (.gst). Click Choose, located the file, then click Open.
The FCS or instrument settings file must be from InCyte™, version 3.0, or later.
• compensation settings – either from an InCyte™ FCS file or a saved instrument
settings file (.gst). Click Choose, located the file, then click Open.
The FCS or instrument settings file must be from InCyte™, version 3.0, or later.
7
Click Adjust Settings.
If you retrieved instrument settings and you wish to skip the adjust settings step, click
Acquire to proceed to acquisition.
A dialog box appears prompting you to load the control sample.
Adjusting Instrument Settings
During adjust settings, if you need to change the sample for a particular step, such as
compensation, click the Next Step button, then Adjust Settings, then load the sample, and
click OK.
4-8
8
Mix a stained cell sample and load it on the easyCyte™ System.
We recommend using a stained negative or isotype control sample for the initial
adjustments.
9
Ensure your sample is loaded and click OK in the load samples dialog.
guava easyCyte™ System User Guide
10 Check the Conc (Cells/µL) value in the Sample Info pane and ensure that it is less
than or equal to 500.
 NOTE: If the value is greater than the high limit for the corresponding flow rate, dilute
the sample with the appropriate buffer to lower the concentration and minimize the
risk of coincident events. For optimal performance, we recommend a concentration
of 250 cells/µL or lower.
11 To fine tune the settings, you can make the following
Compensation Controls
adjustments using the Gain, Compensation, and
Miscellaneous Controls. Open each instrument adjustment
window using the Tools menu or the icons in the tool bar (left
Gain Controls
edge of the application window). If necessary, click the
window’s title bar to drag the window to a new location.
Miscellaneous Controls
• Use the Miscellaneous Controls to set the Flow Rate to
Very Low (0.12), Low (0.24 µL/s), Medium (0.59 µL/s), or High (1.2 µL/s). The
default flow rate is Medium. Set the Refresh Rate to the maximum number of
events (100–5000) you want to display.
 NOTE: If you change the flow rate during the adjust settings step, we recommend
that you repeat the adjust settings step at the new flow rate to ensure that the
markers and threshold are set correctly.
• If you wish to save area measurements for all parameters, select Enable Area
Mode. Area will be acquired for all parameters. The threshold parameter, which is
always height, will appear in height (H) at the bottom of the plot parameters list.
Use the Area/Width menu if you want to acquire width for one parameter.
If you want to acquire area for only one parameter, do not check the Enable Area
Mode check box. Instead, select the parameter from the Area/Width menu. The
area and width for the selected parameter will be acquired. Use the Area and/or
Width Scaling Control sliders to reduce or amplify the signal so that the cells are
visible and on scale.
Select Enable Area Mode to acquire
pulse area for all parameters.
Or, select an individual parameter
from the Area/Width menu to acquire
area and width for that parameter.
• The time parameter is saved automatically. Use the Time Scaling Control slider to
set the maximum time (in seconds) to record. Adjust the y-axis (count) scale on the
plot, if necessary.
guava InCyte™ Sample Acquisition
4-9
• Set up the type of plots and parameters you wish to display. If you retrieved a
Method, the plots are configured with the regions and gates defined in that Method.
Modifications may be necessary to accommodate the new data. For more
information on plots and changing the parameters, see page 4-2.
• Use the Gain Controls to select the Threshold parameter from the drop-down
menu.
• Use the Gain Controls to adjust the gains (using the sliders or the arrow keys on
the keyboard) to a value from 1 to 1024. You can also type a numerical value in any
of the Gain text boxes to adjust the settings. Adjust so that the negative population
(or isotype) is positioned in the lower-left corner of the fluorescence plot and the
cells are evenly distributed in the lower-left quadrant. Start from a lower gain setting
and gradually increase the value. For greater detection and sensitivity across a
broad range of fluorescence, the overall gain range can be adjusted using the High
check box. By default, all boxes are checked. If further adjustment is needed or the
signal is at its maximum, remove the checks from the High Calibration check boxes
and allow the gain to stabilize for 2 to 5 seconds.
 NOTE: Use the Clear Events button in the Sample Info panel to clear the display.
• To acquire 4 log decades, remove the check from the 5 decade Acquisition check
box.
• To acquire 4 log decades, remove the check from the 5 decade Acquisition.
• If the red and/or violet laser is not needed for your application, you can turn it off by
removing the check. For information on the laser and optical filters required to
detect the various fluorochromes, see "easyCyte Lasers and Fluorescent Filters" in
Specifications.
 NOTE: Selecting and deselecting lasers will result in changes in laser power.
Additionally, %CVs may vary slightly depending on the laser mode selected for
acquisition.
• To adjust the threshold, use the slider or click and drag the threshold marker (dotted
red line) up or down the axis of the dot plot displaying the threshold parameter until
the desired amount of debris or other unwanted events are eliminated below the
threshold. You can also enter a numerical value in the text box and press Enter on
the keyboard.
• To adjust compensation, load the appropriate sample, and proceed with the
compensation adjustments. You may want to adjust each compensation setting
individually using single-stained controls.
4 - 10
guava easyCyte™ System User Guide
Use the Compensation Controls to select the radio button for the parameter you
wish to subtract, then use the slider to adjust the percentage of the overlapping
signal to be removed from the detector.
For more details on specific compensation adjustments and examples, see
“Performing Manual Compensation” on page 4-34.
12 You may set regions and gates prior to acquiring the samples. See “Regions, Gates,
and Statistics” on page 4-19” and “Gates” on page 4-22 for information.
If you wish to apply a count gate, create and define the gate, then select it from Count
Gate drop-down menu under Sample Info. You can select the count gate from the
Adjust Settings screen only.
You can also view real-time statistics during the adjust settings step. See “Stat
Markers” on page 4-26 for information on creating stat markers and viewing the realtime statistics.
13 When you are finished adjusting settings, click the Next Step button.
If necessary, you can repeat the adjust settings step to ensure that other samples
(such as another positive control) are on scale, appropriately positioned, and
compensated, by clicking Adjust Settings, loading the sample, and clicking OK.
If you wish to save the instrument settings, click the Save Settings icon in the control
panel. Enter a file name and click Save to save a .gst file. Once the worklist is
complete you can no longer save the instrument settings.
14 Vortex the first sample and load it on the instrument.
15 Enter the number of events to acquire in the Sample Info panel.
The default number of events to acquire is 5000.
guava InCyte™ Sample Acquisition
4 - 11
16 If you want to identify individual samples or sets of samples, enter an optional ID in
the Sample ID field.
The sample ID may be any text up to 40 characters long. If you do not enter a sample
ID, the default sample number will be used as the sample ID, for example, Sample #1,
Sample #2, etc.
 NOTE: If you click Autostart Acquisition and wish to enter a sample ID, type the ID
into the Sample ID field after acquisition is complete and before loading the next
sample.
17 Click the Acquire Next Sample button.
The system acquires the first sample.
 ACQUISITION NOTES
• If you select Autostart Acquisition, acquisition automatically starts when you load
each tube. You do not need to click Acquire Next Sample. Autostart Acquisition
automatically turns off if you click any of the following: Adjust Settings, Retrieve
Settings, Quick Clean, or Backflush. You must recheck the box to continue using
the feature.
• If the acquisition rate appears to slow dramatically,
the fluid pathway may be blocked. Click the Abort
button, then click Capillary Cleaning Tools and
select Backflush. Load a tube of 20% bleach and
click OK. When the backflush is complete, select
Quick Clean. Load a tube of DI water and click
OK. Click Acquire Next Sample to continue.
4 - 12
guava easyCyte™ System User Guide
• The progress bar provides an estimate of the target event count during the
acquisition period, which times out after 1.75 minutes (high flow rate), 3.5 minutes
(medium flow rate), 7 minutes (low flow rate), or 10 minutes (very low flow rate).
 NOTE: If a clog or loss of signal is detected, we recommend removing the clog by
running a Quick Clean with water, then a Quick Clean with ICF, followed by
Backflush into an empty tube, then another Quick Clean with ICF, and a final Quick
Clean with water.
18 Click Save and Close Current Sample.
You may still enter or change the Sample ID for the current sample before clicking
Save and Close Current Sample.
19 Repeat steps 14 through 18 for the remaining samples.
20 Click the Stop and Close Session File after the last sample.
21 When you are finished, click Capillary Cleaning Tools and select Quick Clean. Load
a tube of deionized water and click OK. If you ran blood samples, run two Quick Clean
cycles with water, followed by one cycle using ICF, and finally one cycle using water to
rinse.
At the completion of the run, a copy of the Data, Method, and AnalysedGroup are
automatically loaded into the Analysis control panel. guava InCyte™ saves the data for all
samples as a single FCS 3.0 file to the specified location.
The FCS file contains:
• the acquired data for all tubes in the run
• the Method (plots, regions, gates, and metrics, if applicable)
• an AnalysedGroup (the data paired with the Method)
• instrument settings (gains, compensation, miscellaneous settings)
guava InCyte™ Analysis
InCyte™ Software allows you to open and analyze any guava® FCS 3.0 data file,
regardless of the software module used for acquisition. An analysis-only version of
InCyte™ 3.0 is available for MacOS 10.6 and later. See the readme file for more
information and details on where to go to download the software.
You can proceed to analysis from the acquisition screen.
guava InCyte™ Analysis
4 - 13
The Analysis Panes
Click the Analyse button at the top of the control panel to access the Analyse control
panel. If you click Analyse from the Acquisition screen after acquiring a sample, the data
from the last sample acquired is displayed.
Tool bar
Analysis control panel:
• Data pane
• Analysis Methods pane
• Analysed Data pane
Plate map
Figure 4-2 InCyte™ application window – Analysis screen
The Analyse control panel provides a working outline for each analysis session. It
contains three panes—the Data, Analysis Methods, and Analysed Data. An FCS file
generated using InCyte™ will automatically contain all three elements.
• The Data pane lists all open FCS files, as well as any groups (FCS file subsets)
created. For more information on groups, see “Creating a Group” on page 4-46.
• The Analysis Methods pane contains all Method(s), each comprised of its associated
plots, regions, gates, and metrics. Methods can be thought of as analysis strategies.
• The Analysed Data pane lists all user-defined AnalysedGroups. Analysed Groups are
created by pairing the data file with Method—each consisting of a data file (or group)
with its associated Method. When you close an Analysed Group, the associated FCS
file and Method will close automatically.
Place your cursor over an icon in any of the three panes to see text describing the icon
. For more information on the control analysis panels, see “Analyse
Control Panel” on page 1-19.
4 - 14
guava easyCyte™ System User Guide
Analyzing Files Acquired Using InCyte™
Data files acquired using InCyte™ will already have the necessary components for each
of the three Analysis panes—the data file, the Method, and the Analysed Group. Simply
open the FCS file.
During analysis, the original FCS file and any analysis settings (plots, parameters, gates)
will not be affected. If you wish to save any changes to the newly analyzed InCyte™ file,
simply resave the file using the Analysed Data pane (Save Analysed Group button).
You can proceed to analysis from the acquisition screen.
 NOTE: You can analyze tube-based data but the results cannot be displayed in the
plate map. Additionally, the gates and markers do not carry over from one tube to the
next. Tubes must be analyzed individually (tube-by-tube basis). Use the Stat Setup
feature to obtain results for tube data.
 NOTE: If you open and analyze multiple files within a session, all the FCS data files,
Methods, and Analysed Groups will remain in the panes from all file that were
opened. If they have already been saved, you can remove them from the pane by
selecting the item and clicking the Delete icon at the bottom of that pane.
1
From the Analyse screen, choose File > Open from the menu bar. Select an FCS file
for analysis and click Open. You can also use the Open Group icon in the Data pane
or the Open Analysed Group icon in the Analysed Data pane. You can also drag and
drop an FCS file from a folder to the Data pane.
The data for the first sample appears. For information on selecting the number and
type of plots (dot plot, histogram, or contour plot) you wish to display, and changing
the plot parameters, see “Plots” on page 4-2.
guava InCyte™ Analysis
4 - 15
Click the + to the left of the data file name to display a list of all samples contained in
the file. All wells with acquired data appear as open circles in the corresponding
locations in the plate map.
Clicking on a sample in the Data pane will display the unanalyzed data for that sample
in the plots, however the information in the plate map will be lost. Click on the
Analysed Group to redisplay the information in the plate map. If the data is already
analyzed, click on the individual wells in the plate map to display the analyzed data for
that well in the plots, or use the arrow keys to quickly scroll through the wells,
displaying the data.
NOTE: An icon appears in the upper-left corner of the plate map. It allows you to toggle
between the tube map and plate map. Samples from tubes acquired on your
easyCyte™ System will always appear in the plate map. If the data set was acquired on
an easyCyte™ HT System, which allows you to acquire from both tubes and a
microplate, the samples acquired from tubes will show up in the tube map.
If applicable, click icon to toggle
between plate map and tube map.
 NOTE: If you wish to create groups of samples for analysis, see “Creating a Group” on
page 4-46.
2
4 - 16
You are now ready to start customizing your Analysis Method using regions and
gates. See “Regions, Gates, and Statistics” on page 4-19.
guava easyCyte™ System User Guide
Analyzing Files Acquire Using Other guava® Software Modules
Data acquired using any guavaSoft™ module other than InCyte™, for example,
ExpressPro, will not have an Analysis Method associated with it. However, when you
open the file in InCyte™, a default Method will automatically be created.
1
From the analysis pane, choose File > Open from the menu bar. Select an FCS file
for analysis and click Open. You can also use the Open Group icon in the Data pane
or the Open Analysed Group icon in the Analysed Data pane. You can also drag and
drop an FCS file from a folder to the Data pane.
The data for the first sample well appears. For information on selecting the number
and type of plots (dot plot, histogram, or contour plot) you wish to display, and
changing the plot parameters, see “Plots” on page 4-2.
Click the + to the left of the data file name to display a list of all sample wells and
tubes contained in the file. All wells with acquired data appear as open circles in the
corresponding locations in the plate map. Data for any well can be displayed in the
plots by selecting the sample from the file list or clicking on a well in the plate map.
guava InCyte™ Analysis
4 - 17
2
When a data file is generated in a guava® software module other than InCyte™,
InCyte™ will automatically create a default Analysis Method and Analysed Group for
the data file/set. The Method will contain a percent (%) metric, initially. If you wish to
open an existing Method use the Open Method icon. Method files have the extension
.gsy. To rename the Method, double-click it and enter a new name related to this
analysis.
Analysis Method containing a metric, gate list, and
region list.
Click New Method to create a new Method.
3
If you opened an existing Method, you will need to drag it to the Analysed Group.
Data and Method are
automatically paired
to create an Analysed
Group. If you open a
saved Method, you
will need to drag it to
the Analysed Group.
Notice the sample wells in the plate map become dark blue (if the Method was newly
created) or various shades of blue (if a previously defined Method is used). You can
place the cursor over any well in the plate map to view results for that sample.
Clicking on a sample in the Data pane will display the unanalyzed data for that sample
in the plots, however the information in the plate map will be lost. Click on the
Analysed Group to redisplay the information in the plate map. Click on the individual
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guava easyCyte™ System User Guide
wells in the plate map to display the analyzed data for that well in the plots, or use the
arrow keys to quickly scroll through the wells, displaying the data.
 NOTE: Any data file or Method file can be replaced by simply selecting it from the
respective pane and dragging to the Analyzed Group. This allows you to quickly
interchange data files and Methods.
AnalysedGroup containing a data file and Analysis Method.
To save an AnalysedGroup, click on it in the Analysed Data pane to select it, then
click the Save Analysed Group icon. Navigate to the location, enter a name, and click
Save. The AnalysedGroup will be saved as an FCS file and will contain the method
(regions, gates, metrics, and stats), as well as the data and the instrument settings.
4
You are now ready to start customizing your Analysis Method using regions and
gates. Refer to “Regions, Gates, and Statistics” on page 4-19.
Regions, Gates, and Statistics
Regions
InCyte™ offers six types of regions/markers—ellipses, rectangles, octagons, polygons,
and quadrant markers for dot plots, and histogram markers for histograms. All of these
regions are created using either the New Region icon in the plot tool bar (see “Plots” on
page 4-2), except the quadrant marker, or the New Stat Marker icon in the tool bar, which
allows you to select statistics for the region/marker. For information on creating
quadrants, refer to “Stat Markers” on page 4-26. A single Analysis Method can contain a
maximum of 24 regions or stat markers, and you can have multiple stats per marker.
InCyte™ regions are associated with the one (histogram) or two (dot plot) parameters in
which they were created. If you change the x- or y-axis parameter on the plot, the region
is no longer displayed. However, it is still contained within the Method.
1
Once an AnalysedGroup is created, either by pairing a data file with a Method or
dragging the AnalysedGroup to the legend, click the New region icon in the plot tool
bar and select a region from the pop-up menu.
You are prompted to enter a region name. The default is R1. When creating a
polygon, this dialog appears after you create the region.
Regions, Gates, and Statistics
4 - 19
2
Enter a name for the region and click OK.
You may leave it R1, but a unique region name can be helpful in differentiating
Analysis Methods.
The newly created region appears on the plot with the region name. Click and drag
the name to move it. The region is listed in the Region List under the Method (see
below) and in the Region List table (Region List icon in the main tool bar or Tools >
Show Region List). To delete a region, see “Region List” on page 4-21.
3
Adjust the region to encompass the data. Or, for a polygon, create the region.
• To adjust an elliptic region, click anywhere on the edge
of the ellipse, except on a handle, and drag it to a new
location. The ellipse has two handles. The open circle
allows you to narrow/widen the ellipse. The solid circle
allows you to lengthen, as well as rotate the ellipse
around a point opposite the solid circle.
• To adjust a rectangle region, click anywhere on the
edge of the rectangle, except on a handle, and drag it to
a new location. The rectangle has five handles. The
open squares allow you to extend at the corresponding
corner. The solid square allows you to rotate and resize.
• To adjust an octagonal region, click anywhere on the
edge of the octagon, except on a handle, and drag it to
a new location. The octagon has eight handles. Click
and drag the handles to adjust the shape of the
octagon. To add or remove segments, right-click on a
handle and select Insert Line Segment or Delete Line
Segment.
• To create a polygon region, click to add the first
handle, then move the cursor to the next point and click.
Continue creating up to 32 segments until the shape is
complete. Finish by clicking again at the first handle to
close it. To move it, click anywhere on the edge, except
on a handle, and drag it to a new location. To adjust the
shape, click and drag the handles. To resize or rotate,
press the Shift key and click and drag a handle.
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guava easyCyte™ System User Guide
• To adjust a histogram region, click either of the two
handles. The histogram marker can be moved vertically
as well as horizontally.
Region List
When you create a region, it appears in the Region List. To
display the list, click the Region List icon in the tool bar or choose Tools > Show
Region List. The Region List displays a list of all regions and quad stat markers (see
page 4-26) in the selected Analysis Method, their type, and plots axes.
• To display all the columns in the Region List window, click Resize.
• To delete a region from the plot, click to select it, then right-click and select Delete
Region. You can also select the region in the Region List window and click Delete.
Click OK in the confirmation box. The region will be deleted from all associated
plots. The region name in the Analysis Method will appear as ???.
Global and Local Region Adjustments
You can make adjustments to regions for specific samples within a data set. For example,
you may wish to tweak a region for the data in one or more samples to compensate for a
shift in the data.
1
To change a region for an individual sample, press and hold the Ctrl key while
manipulating the region (elliptic, rectangle, octagon, or histogram).
To change the region for multiple samples, highlight the desired wells, then hold the
Ctrl key while manipulating the region. The change will be applied to all the
highlighted samples.
The region will turn from red to black, indicating that it is a local region—it applies only
to the sample data displayed in the plots.
 NOTE: If you manipulate a global region without holding down the Ctrl key, the
change will automatically apply globally (to the data for all samples in the run).
Once a local region is created:
• if you have only one local region in the data set and you attempt to manipulate it
without holding down the Ctrl key, the change will become global and apply to all
regions in the data set.
Regions, Gates, and Statistics
4 - 21
• if you have multiple local regions and you attempt to manipulate a single local or
global region without holding down the Ctrl key, the following message appears:
• To apply the change to the region you are adjusting and all global regions, but
keep any other local regions unchanged, click Keep Local.
• To apply the change to all samples in the run, click Make Global.
• To cancel the change and not apply it to any regions, click Cancel.
If you want to make an adjustment to a local region to manipulate it further, hold down
the Ctrl key to keep the change local and not affect any other regions—local or global.
After making a region(s) local, you can still make adjustments to global regions
without holding down the Ctrl key. When the dialog box appears, click Keep Local.
The adjustment will apply to all global regions. The local regions will remain local and
unchanged.
Gates
A gate can be as simple as a single region, or a complex combination of regions and
multiple parameters. Gates allow you to further characterize and isolate populations
based on the regions. A maximum of 32 gates can be created for one Analysis Method.
You can create gates for analysis and/or acquisition. Count gates used during acquisition
allow you to acquire specific populations of interest. All events above the threshold are
saved to the file whether they are in the gate or not. However, the number of Events to
Acquire is applied to events that fall within the gate.
Defining a Gate
The gating process has been simplified by dragging and dropping regions. Any region
can be dragged from one plot to any other plot. This process allows you to create gates
defined with the “AND” operator. You can also create gates using the OR and NOT
operators. Refer to “Gate List” on page 4-24.
1
4 - 22
Click to select the region (handles appear), then click in the center of a region and
drag it to another plot.
This “applies” the data within the boundaries of the region to the second plot. For
example, if you created a region on live cells in plot 1 and drag the live-cell region to
plot 2, only the live cells will be displayed in plot 2. A New Gate Name dialog box
guava easyCyte™ System User Guide
prompts you to rename the newly created gate. You can enter a more meaningful
name or use the default.
 NOTE: You can also define a gate by typing the regions and the operators AND,
OR, and NOT. Refer to “Gate List” on page 4-24.
If the Analysis Method has gates already defined, you can also select a gate from the
Plot gate menu in the plot tool bar.
Select the region, then drag it from one
plot to another to apply a gate.
When you apply a gate, the following changes occur:
• The events displayed in Plot 2 will automatically change to reflect only those
defined by the region that was dragged in. To view the ungated data again, click
the Plot gate icon to the right of the plot and select <ungated>.
• The color of the newly gated data will reflect the color of the gate defined in the
Gate List window. The default color for the first gate is red.
• The plot heading will reflect the gate applied (for example, Plot P01, gated on
P02.R1).
• The newly created gate will appear in the Analysis Methods pane under the
Method, in the Gate List (see "Gate List" below), and the Plot gate pop-up menu to
the right of the plot.
 NOTE: Changes made to the position or size of any region(s) used to define a gate
will automatically be reflected in all plots gated by that region.
Regions, Gates, and Statistics
4 - 23
Gate List
When you create a gate, it appears in the Gate List. To display the list, click the Gate List
icon in the tool bar or choose Tools > Show Gate List. The Gate List displays a list of all
gates in the selected Analysis Method, their definition, and color.
• To display all the columns in the window, click Resize.
• To delete a gate from the list, select the gate and click Delete. Click OK in the
confirmation box.
• To change the gate color, double-click the color in the Color column. The
ColorPickerDialog appears. See “Changing the Color of Gated Data” on page 4-25
for information on using this dialog box to change the color.
• You can turn a region into a gate by entering a name for the gate in the Name field
and typing the region name (for example, R1) in the Definition field.
• You can also define a gate using the operators AND, OR, and NOT. Create regions
to identify the subpopulations of interest. Open the gate list and type an
appropriate name for the gate. Type a definition, for example:
– R1 AND R2 means the event must be in both the R1 and R2 regions to be
included in the gate.
– R1 OR R2 means the event must be in either the R1 or R2 region to be included
in the gate.
– R1 AND (NOT R2) can be used if R1 and R2 overlap and you want to include
events in R1 but not in R2.
The gate can now be applied to any plot by clicking the Plot gate icon to the plot
tool bar and selecting the gate from the list.
• The ColorGate box allows you to view a backgate. Create a gate in one plot, then
drag that gate to the plot of interest. This applies the gate to the second plot and
shows only the gated events. Select Tools > Show Gate List and check the
ColorGate box for the gate. The gated events appear in both plots in the selected
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guava easyCyte™ System User Guide
color. View the second plot without a gate (select <ungated> from the plot tool bar)
to see the color gated events in relationship to all events.
A lymphocyte gate was created in the RED (CD3 PE-Cy5.5) vs SSC plot, then dragged to the YEL (CD14 PE)
vs GRN (CD15 FITC) plot. Granulocyte and monocyte gates were created in the YEL vs GRN plot and
dragged to the RED vs SSC plot. ColorGate checkboxes were selected for all gates to apply color to the
gated events. All plots are viewed ungated to show all data and gated data in colors.
Changing the Color of Gated Data
You can change the color of data within a gate.
Open the Gate List and double-click the color in
the Color column to open the ColorPickerDialog.
Use this dialog to change the color, hue,
saturation, and brightness.
1
To change the color, click the RGB button and
use the sliders to adjust each color. Or, click to
select a color in the outer circle. Drag the
pointer to change the color.
2
To adjust the hue, saturation, or brightness,
click the HSB button and use the sliders to
make the appropriate adjustments. Or, click a
shade in the inner triangle.
3
Click OK when you are finished.
Regions, Gates, and Statistics
4 - 25
Stat Markers
The Stats feature allows you to derive, display, and export statistics for single samples,
groups, or whole data sets. Each stat is derived from a region/marker. Stats can be
assigned to existing regions, or new regions can be created. A single Analysis Method
can contain a maximum of 24 regions/markers.
1
Click the New Stat Marker icon in the plot tool bar and select the appropriate option.
You can obtain statistics for any existing regions in the plot. Or, you can create a new
region and obtain statistics for that region.
• Select New Elliptic/Rectangle/Octagonal/Quad/Histogram Stat Marker to add a
new region and obtain stats for it. Histogram Stats appear only in a histogram plot.
• Select New Stat for R1 (or name of existing region) to obtain stats for an existing
region.
• Select New Expression Stat to create an expression statistic. See “Creating
Expression Statistics” on page 4-29.
• Select Show Stat Setup to display the Current Run Stats. See "Current Run Stats
Window" in the following section.
• Select Edit Plot Corner Labels to add custom labels to your plots. See “Labeling
Plot Corners” on page 4-31.
The Add New Stat Marker window appears for the stat type you selected. The default
base stat name is the same as the gate name. Base stat implies it is the base name
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guava easyCyte™ System User Guide
before you selected a specific metric (stat), then the stat name becomes
BaseStatName.Metric.
If a stat with the default name already exists, the field
will appear red. You must enter a new stat name.
2
Select the metric—Percent, Count, Mean, and Median, Concentration, or %CV.
3
For quad stats only, select the quad region(s) of interest. Select Show on plot if you
wish to see the stat for each quadrant appear in the corners of the plot. If multiple
parameters are selected, the count appears by default.
Quadrant markers will appear on the plot after you click OK.
4
Click OK.
The Current Run Stats window (see below) appears with the newly created stat. The
value listed corresponds to the sample well highlighted in the plate map. Each stat is
automatically applied to all samples in a given data set. To see the results for any well,
click on the well in the plate map. If you adjust the marker/region or any region that is
part of the gating strategy, the statistics are automatically updated.
To adjust quadrant markers, click to select the markers.
The handles will appear solid. Position the cursor over
the handle at the intersection and drag to the desired
location. You can adjust the angle of the markers ±44°
from their original locations. Drag the handle (solid
circle) towards the end of the marker and tilt it to the
desired location.
Regions, Gates, and Statistics
4 - 27
Current Run Stats Window
The Current Run Stats window appears when you create a stat. You can also click the
Show Current Run Stats icon in the main tool bar or select Tools > Show Current Run
Stats from the menu bar to display the window. For each Analysis Method, all stats for all
plots are listed in this window. The Values displayed correspond to the sample well
highlighted in the plate map. Simply click any well to see the value for that sample. Stats
are listed in the order they were created. To reorder them in the list, see “Current Run
Stats Setup Window” on page 4-28.
For each Analysis Method, all stat markers are listed in the Region List, as well as the
Analysis Methods pane. All stats are saved with the Method.
customize stats
• Click Setup to customize the stats listed in the Current Run Stats window.
• Click Print Stats to print the statistics listed in the table.
• To copy the window to the clipboard right-click in the window and select Copy to
Clipboard.
Current Run Stats Setup Window
To customize the statistics that are listed in the Current Run Stats window, click Setup.
The Current Run Stats Setup window appears.
Click the box to the far left and
drag the row up or down in the list.
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guava easyCyte™ System User Guide
Click to remove a column from the
Current Run Stats window.
• To change the Stat Name, double-click the name and enter a new name. To change
the metric, gate, quadrant, marker name, or parameter, double-click the item and
select the new one from the drop-down list. Expression stats cannot be modified. They
can only be deleted.
 NOTE: If you wish to change the parameter, double-click to display the parameters
•
•
•
•
for that plot. If you wish to see all available parameters for the data set, press Ctrl
and double-click the parameter value. For a parameter to be displayed, you must
select it in the Add New Stat Marker dialog.
To remove a column, remove the “Show” check mark for the selected parameter(s)
and click Done. The Current Run Stats window is updated.
You can also create stats directly from the Current Run Stats Setup window. Click Add
New Stat to display the Add New Stat Marker window. This window is similar to the
window that appears when you select Show Stat Setup from the New Stat Marker
icon in the plot tool bar (see page 4-26). However, this stat marker window allows you
to create a stat independent of a plot and based on any gate. Enter a stat name, then
select the marker/region, gate, parameter, and metric, and click OK.
To reorder the rows and/or columns in the Current Run Stats Setup window, click the
far-left box of the row and drag the row up or down, or click the column header and
dragg the column to the left or right. You cannot move the Stat Name or Value column.
They are fixed.
To delete a stat, select the stat from the list and click Delete.
Creating Expression Statistics
Expression stats allow you to create custom statistics. For example, you can apply the
dilution factor to a count or concentration value of a specific population to get the actual
value for your diluted sample. Or, you can create an expression statistic that adds the
events in two quadrants of quad markers.
1
Create statistics as you normally would.
2
Click the New Stat Marker icon in the plot tool bar and select New Expression Stat.
3
Enter a stat name for the expression.
Regions, Gates, and Statistics
4 - 29
4
Right-click in the Expression area of the window and select the stat. Type an operator
(+, –, *, /, or ^ for exponentiation). Complete the expression.
 NOTE: To create an expression for a specific tube, press Ctrl and right-click the
Expression area. The pop-up menu allows you to select a specific tube.
The following example shows an expression created to apply the dilution factor of 20
to events in the upper-left quadrant of a plot.
Right-click and select the statistic to build the expression.
Enter the appropriate operator, and continue building
expression.
4 - 30
5
Click OK.
The expression appears in the Current Run Stats window.
6
Click the Show Sample Info icon in the tool bar to the left of the data panes to open
the sample information window. Enter the dilution factor for the sample and press
Enter.
guava easyCyte™ System User Guide
7
The updated concentration value, based on the new dilution factor, appears in the
Current Run Stats window.
Labeling Plot Corners
You can add custom labels to the corners of any plots. Default labels already appear in
plots with quadrant markers, if you select Show on plot when setting up the quadrant
using the New Quad Stat Marker option.
1
Click the New Stat Marker icon in the plot tool bar and select Edit Plot Corner
Labels.
The Edit Plot Corner Labels dialog appears.
Labels for quadrants.
2
If you are adding labels to a plot with quadrant markers, click the default [count] label
and type a new label. If you are adding labels to a plot without quadrant markers,
simply type the label in the corresponding box.
3
Click OK. The labels appear in the corners of the plot.
Regions, Gates, and Statistics
4 - 31
Group Stats Window
The Group Stats window displays the statistical values for all samples in the data set (or
group). To display the Group Stats window, click the Show Group Stats icon in the main
tool bar or select Tools > Show Group Stats from the menu bar.
• Click Setup to customize the stats listed in the Group Stats window.
• Click Export To CSV to export the group stats to a comma-separated values file for
analysis using a spreadsheet program. Select the location, enter a file name, and click
Save.
• Click Print Stats to print the statistics listed in the table.
• To copy the window to the clipboard, right-click in the window and select Copy to
Clipboard.
Group Stats Setup Window
To customize the statistics that are listed in the Group Stats window, click Setup. The
Group Stats Setup window appears. To remove a column, remove the “Show” check mark
for the selected parameter(s) and click Done. The Group Stats window is updated.
You can reorder the rows by clicking the far-left box of the row and dragging the row up or
down.
To reorder the rows, click the box
to the far left of the row and drag
the row up or down in the list.
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guava easyCyte™ System User Guide
Exporting Results
Exporting guava InCyte™ Results to a Spreadsheet File
For each AnalysedGroup, you can export the results to a comma-separated values (CSV)
file for analysis using a spreadsheet program such as Microsoft Excel. The CSV file will
contain the following statistics: Group name, Stat Type, Parameter, Stat gate, Control
gate, Log Display, Max, Threshold Max%, Threshold Max, Threshold Min%, Threshold
Min. In addition, each sample well will include a derived statistical value, its unit of
measurement, a control well (if applicable), the well's sample ID, and the FCS 3.0 file
from which it was derived.
1
Right-click on any well and select Export Heatmap Stats to CSV.
If the HeatMap legend is divided into multiple sectors, then data from each sector will
be exported. If you wish to export results for a single sector (or AnalysedGroup), first
click on the specific sector in the legend, then right-click the well and select Export
Heatmap Stats to CSV.
To export the group stats to a CSV file, refer to “Group Stats Window” on page 4-32.
 NOTE: You can copy and save any of the plots, HeatMap, or IC-50 graphs by rightclicking, then selecting Copy To Clipboard.
Exporting guava InCyte™ Results to FCS and List-Mode Files
You can export guava InCyte™ results from the current run or any sample run to both
FCS 2.0 and FCS 3.0 files. The data must have an AnalysedGroup associated with it.
One FCS file is saved for each sample acquired. You can analyze both FCS 2.0 and 3.0
files using a third-party flow cytometry analysis application. FCS 3.0 files are exported
with all parameters included. FCS 3.0 files with semi-automated or post-acquisition
compensation may be compatible with some third-party applications. For more
information, contact EMD Millipore Technical Support. You can also export list-mode data
files.
1
To export analysis results for all sample data within the run to individual FCS 2.0 or
3.0 files, select File > Export to FCS 2.0, Export to FCS 3.0, or Export List Mode
Data.
You can also select a specific AnalysedGroup from the Analysed Data Pane and
select the appropriate export option from the File menu.
2
Select the folder where you want to save the file, and enter a file name. Click Save.
The samples are numbered sequentially and a number is automatically appended to
the file name. For example, if the sample number is A01, the file will be named
filename-1.FCS, A02 is filename-2.FCS, etc.
Regions, Gates, and Statistics
4 - 33
Compensation
This section provides details on specific compensation adjustments for your stained
samples during acquisition. It also includes information on performing post-acquisition
compensation and automated compensation on data files acquired with ExpressPlus,
ExpressPRo, and InCyte™ only.
Performing Manual Compensation
You can perform compensation before or after acquiring your samples. Use the
Compensation Controls to select the radio button for the signal you wish to subtract, then
use the slider to adjust how much of that signal to remove from the detector/channel.
Click the Show Compensation Controls icon in the tool bar to open the Compensation
Controls window.
 NOTE: The controls at the far left are for semi-automated compensation only.
You must check the compensation for each fluorochrome combination you are using.
Compensation settings are correct when the center of the stained population is aligned
with the center of the isotype or negative control population. Avoid having too many cells
touching the axis (overcompensated).
 NOTE: Some fluorochromes require very little compensation because they have little
overlap, such as PE into the GRN channel (GRN-B – % YEL-B), as shown in the first
three plots below; whereas others require much more compensation because they
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guava easyCyte™ System User Guide
have more overlap, such as FITC into the YEL channel (YEL-B – % GRN-B), as
shown in the last three plots below.
Correct Compensation
Over Compensation
GRN-Log
No Compensation
YEL-Log
GRN–%YEL– FITC Single-color stained cells
Correct Compensation
Over Compensation
GRN-Log
No Compensation
YEL-Log
YEL–%GRN – PE Single-color stained cells
During Acquisition
1
Select the radio button for the signal you wish to subtract, then use the slider to adjust
the percentage of the signal you wish to subtract from the detector.
For example, to remove the FITC signal from the YEL detector, select the –% GRN-B
radio button under Dye. Then adjust the YEL slider to remove the FITC signal from
the YEL channel (YEL-B – % GRN-B). Next, using the PE single-color control, select
the –% YEL-B button, and adjust the GRN-B slider to remove the PE signal from the
GRN channel (GRN-B – % YEL-B).
Post-Acquisition Compensation
InCyte™ allows you to perform post-acquisition compensation on data files acquired using
software modules that have the compensation feature and allow you to adjust
compensation during acquisition, such as ExpressPlus, ExpressPro, and InCyte™.
 NOTE: You can adjust compensation only; you cannot adjust gain settings.
Compensation
4 - 35
1
Open the FCS file from the Analyse control panel and set up the plots.
We recommend using single-color samples, then checking the compensation again
with a sample stained with all fluorochromes to fine-tune, if necessary.
2
Select the radio button for the signal you wish to subtract, then use the slider to adjust
the percentage of the signal you wish to subtract from the detector.
For example, to remove the FITC signal from the YEL detector, select the – % GRN-B
radio button under Dye. Then adjust the YEL slider to remove the FITC signal from
the YEL channel (YEL-B – % GRN-B). Next, using the PE single-color control, select
the –% YEL-B button, and adjust the GRN-B slider to remove the PE signal from the
GRN channel (GRN-B – % YEL-B).
3
Repeat for the remaining compensation adjustments for your samples.
 NOTE: The controls at the left of the window are for semi-automated compensation
only.
Settings are applied to all samples in the data set, as well as all groups derived from
the data set. Adjustments made to a sample within a group, are applied to all samples
in the originating data file.
Performing Semi-automated Compensation
InCyte™ allows you to perform semi-automated compensation on data files acquired using
software modules that have the compensation feature and allow you to adjust compensation
during acquisition, such as ExpressPlus, ExpressPro, and InCyte™. You must have
acquired single-color control samples to use the semi-automated compensation feature.
 NOTE: You can adjust compensation only; you cannot adjust gain settings.
4 - 36
1
To adjust compensation, open the FCS file from the Analyse control panel and set up
the plots.
W recommend using single-color samples, then checking the compensation again
with a sample stained with all fluorochromes to fine-tune, if necessary.
2
Click the Show Compensation Controls icon in the main tool bar to open the
Compensation Controls window.
This window is specific for compensation during analysis. It contains a slider for each
parameter and a list of controls in the far left panel. These controls correspond to
each parameter and the fluorochrome used:
guava easyCyte™ System User Guide
Comp Color
Channel
Laser
BLU-V
GRN-V
YEL-V
405 nm (Violet)
RED-V
GRN-B
YEL-B
RED-B
488 nm (Blue)
NIR-B
RED-R
NIR-R
642 nm (Red)
negative or isotype control
 NOTE: If it is necessary to gate the data, pair a Method with the data file to create an
Analysed Group and set regions and gates. Refer to “Gates” on page 4-22.
Otherwise, it is not necessary to create an Analysed Group to adjust post-acquisition
compensation.
3
Click Setup in the Compensation Controls window.
The application screen changes to display two dot plots on the left with the plate map
below, and up to six histograms for each fluorescence channel.
4
Adjust the default region in plot 1 so that it encompasses the events of interest.
5
Drag the isotype (or negative) control from the plate map to the black square at the far
left in Compensation Controls window
6
Drag the next control, for example the GRN control (FITC-positive control) from the
plate map to the green square at the far left in the Compensation Controls window
(see example in figure below).
Compensation
4 - 37
For this example, the green square turns into a circle and the FITC control data is
displayed in the GRN histogram.
7
Adjust the GRN histogram marker to include the positive population.
Dra
g
8
Drag the next control, for example YEL control (PE-positive control) to the yellow
square. Continue dragging the remaining controls to the corresponding Controls
squares and adjusting the markers to include the positive population.
9
Check that the markers are correct for the isotype control.
10 Click Auto. The software automatically calculates the compensation values for each
parameter.
Settings are applied to all samples in the data set, as well as all groups derived from
the data set. Adjustments made to a sample within a group are applied to all samples
in the originating data file.
11 If you need to adjust compensation further, refer to “Post-Acquisition Compensation”
on page 4-35.
12 Click Finish to return to the analysis display.
HeatMap
HeatMapping provides a colorimetric representation of comparative results at the
experiment level. Each Analysed Group is represented by an individual sector in the
HeatMap legend. After the completion of sample analysis, individual parameters can be
compared over the entire experimental run. A visual representation of the data and the
results will appear in the plate map. Well-to-well variations in blue (dark blue = maximum
4 - 38
guava easyCyte™ System User Guide
value, white = minimum value) are based on relative differences in the data as measured
on a linear scale.
The plate map can simultaneously display results for up to six Analysed Groups—the
same number of sectors that the legend is displaying. Place the cursor over any well to
see the result for that Analysed Group. When the HeatMap legend displays more than
one sector, the results for every sector will appear within the well pop-up.
 NOTE: If the Analysis Method used to create the Analysed Group is new (no regions
and/or gates have been defined), the plate map will initially display all wells as dark
blue. Perform the analysis on the data before proceeding to HeatMapping.
One sector in HeatMap Legend
Six sectors in HeatMap Legend
Creating a HeatMap
The following examples show how to use the HeatMap to get a visual and numerical
representation of results for a single or multiple parameters from one data file. This
example shows a sample stained to detect mitochondrial membrane potential, cells
undergoing apoptosis, and dead cells.
Single-Parameter HeatMap Using a Region or Gate
1
Open the Analysed Group.
HeatMap
4 - 39
You can also open an FCS data file and perform the analysis for the populations you
are interested in. See “guava InCyte™ Analysis” on page 4-13, for information.
Jurkat cells were stained with FlowCellect MitoDamage kit to look at mitochondria membrane potential
(MitoSense Red), apoptosis (Annexin V CF488), and cell death (7-AAD). R1 was created for cell
population, then applied to plots 2-4. Three regions were created for each population of interest.
2
Select the gate of interest, then click and drag it to the Analysed Group in the
Analysed Data pane. Click OK to accept the new gate name, or change the name.
As you start to drag the gate, a gray box appears at the cursor. Drag the gate to the
Analysed Group and a plus sign (+) appears on the cursor. Drop the gate in the
Analysed Group.
 NOTE: You can also use the HeatMap legend by dragging the Analysed Group and
the gate to the legend. Then, to update the analysis, simply drag a new gate to the
legend. The plate map will update automatically.
4 - 40
guava easyCyte™ System User Guide
After the gate is applied to the Analysed Group, the wells will change to varying
shades of blue, depending on the population percentages. Place the cursor over any
well to see value for that well.
gg
Dra
ate
MitoSense Red region is dragged to the Analysed Group. Plate map displays well-to-well variation of
relative percentage of mitochondrial membrane potential among samples.
3
To change the metric and/or threshold, see “Metrics” on page 4-43 and “Thresholds”
on page 4-43.
4
To save the HeatMapped Analysed Group, select it and click the Save button (3rd) in
the pane. Navigate to the location where you wish to save it, enter a file name, and
click Save. The Analysed Group will be saved as an FCS file and will contain the
Method (regions, gates, metrics, and stats), as well as the data and the instrument
settings.
Multi-Parameter HeatMap Using a Region or Gate
The following example is the same as the single-parameter HeatMap shown above. In
this example, three Analysed Groups are created to view and compare the results for
three different populations simultaneously.
1
Open the Analysed Group.
2
To compare multiple parameters from one FCS file, duplicate the Analysed Group.
Select the Analysed Group, then click the Duplicate button (4th button) in the pane.
For this example, the Analysed Group was duplicated twice to obtain results on three
populations.
3
Select the first Analysed Group, then click to select and drag the gate of interest to
this Analysed Group. Click OK to accept the new gate name, or change the name.
HeatMap
4 - 41
4
Select the second Analysed Group, then click to select and drag the next gate of
interest to this Analysed Group.
5
Repeat by dragging the remaining gates to the Analysed Groups.
6
(Optional) To change the name of an Analysed Group, double-click it and type the
new name.
7
Click the Pie Legend icon in the tool bar on the left side of the application window to
display the HeatMap legend. Select the number of sectors (N) to display.
8
Click and drag the Analysed Groups to the appropriate sectors. Then, click the center
circle in the legend to display the results for all populations of interest in the plate
map.
 NOTE: When multiple sectors are displayed, no data is shown in the plots and you
cannot modify any Methods (add regions, move gates, etc). To modify individual
Methods, click on the sector you wish to update.
For more information on the HeatMap legend, see “Pie Legend” on page 4-5.
9
The plate map shows each well with the number of sectors used for analysis. Place
the cursor over any well to see all the values for that well.
10 (Optional) To clear a sector, right-click on the sector in the HeatMap legend and select
Clear sector.
11 To change the metric and/or threshold, see "Metrics" and “Thresholds” on page 4-43.
12 To save the HeatMapped Analysed Groups, select one and click the Save button (3rd)
in the pane. Navigate to the location where you wish to save it, enter a file name, and
click Save. The Analysed Group will be saved as an FCS file and will contain the
Method (regions, gates, metrics, and stats), as well as the data and the instrument
settings. Repeat for the remaining groups.
4 - 42
guava easyCyte™ System User Guide
Single-Parameter HeatMap Using a Statistic or Expression Stat
The following example shows how to use the HeatMap to get a visual and numerical
representation of results for a single or multiple parameters from one data file.
1
Open the Analyzed Group.
2
If necessary, create a stat region or a statistic for any region created. For more
information on creating statistic gates and statistics for regions see “guava InCyte™
Analysis” on page 4-13.
3
Click the Pie Legend icon in the tool bar on the left side of the application window to
display the HeatMap legend.
4
Select the statistic of interest, then click and drag it to the Analyzed Group in the
Analyzed Data pane. Click OK to accept the new gate name, or change the name.
Thresholds
The Workspace contains a dual-barred threshold tool for setting upper and lower limits on
statistical values. The threshold bars can be used to set limits on a single AnalysedGroup
at a time (the one that is displayed in the plate map). If the HeatMap is displaying multiple
sectors, the threshold feature is disabled.
Above the threshold panel is the maximum derived value for a
given data set. Each slider tool is based upon a % scale where the
minimum value (0 is lower limit) is Zero and the maximum value
(100 in upper limit) is defined by the maximum value derived from
the analysis. Simply slide the bars up or down to set boundaries for
data display.
max
value
In addition, any well can be selected to represent the MAX value.
To set a MAX, right-click on a well and select Use as Max from the
menu. The new max value will also be displayed at the top of the
threshold panel.
Metrics
Metrics are the statistical parameters applied to a set of gated events. Applying a metric
results in the calculation of a numerical value for each well/sample. These values are
compared across a given data set and determine the well-to-well variability in color
displayed in the plate map. There are six metrics from which to choose:
• Percent (%) - Percentage of events within a given gate relative to the total number of
events displayed within the originating plot.
• Count - Number of events within a given gate.
• Concentration - Event count for a gate as a function of the volume of sample acquired
(events/uL).
• Mean - Mean fluorescent intensity (MFI) for a set of gated events. If derived from a dot
plot, the mean value applies to the parameter associated with the x axis. (For log
scale, the mean is geometric; for linear scale, the mean is arithmetic.)
HeatMap
4 - 43
• Median - Median fluorescence for a set of gated events. If derived from a dot plot, the
median value applies to the parameter associated with the x axis.
• Mean Ratio - A measure of the fluorescence intensity for a set of gated events as
defined by the following: ratio = MFIwell X/MFIcontrol well. The control MFI may be
derived from a single sample or represent the average value from a set of control
samples. To apply a mean ratio, select Mean Ratio. Using the plate map, select the
appropriate control well(s) and drag to the corresponding legend sector. This well’s
value is used as the denominator in the above equation. You must select a control well
as the denominator to get a mean ratio.
Changing the Metric
The default metric for a new Method is percent. To select a different metric, double-click
the metric in the Analysis Methods pane, then open the drop-down list to display the
Metric list. Select a metric to apply it as the statistical parameter for that Method. Once
selected, the newly chosen metric will replace the previous one. If you are heatmapping
and the corresponding Method occupies a legend sector, the plate map will automatically
update to reflect the change.
To change the metric, double-click the metric in the Method and select the new
metric.
IC-50/EC-50
Understanding the kinetic characteristics of a given compound's mode of action is
important for determining efficacy, toxicity, and dosage. Assays measuring parameters
such as apoptotic status or protein expression are commonly used to obtain this
information. Use the IC-50 application to create curves based on the relationship between
concentration and response, where response is measured by changes in the statistical
parameter for the applied Analysis Method. IC-50 (or EC-50) values are derived directly
from the calculated curves.
• The EC50 value is the concentration of a drug, antibody, or toxicant which induces a
response halfway between the baseline and maximum following a specified exposure
time.
• The IC50 is a measure of the effectiveness of a compound to inhibit or reduce a
measurable response by 50% relative to the baseline (which in this case is a
maximum value) and absolute minima.
4 - 44
guava easyCyte™ System User Guide
Creating an EC-50 or IC-50 Curve
1
To determine the EC50 value for a given compound, pair a data file with a Method,
then define the regions and gates.
If the compound-specific sample set is part of a larger data set, create a group to
define this specific subset (see “Creating a Group” on page 4-46). Then drag the
group to the legend sector replacing the original FCS data file.
2
Click the Show Pie Legend icon in the tool bar to open the workspace. Click IC50 at
the top of Workspace.
A graph appears where x and y axes are defined as Concentration (log10 scale) and
response level % (linear), respectively.
The y coordinates correspond to the numerical values derived from the
AnalysedGroup. Values are converted to a relative scale where the maximum derived
value is set to 100% on the y axis.
The x coordinates represent the actual compound concentrations used at each step
in the titration. The axis display is open and set automatically from the range of
concentrations used.
3
To set the x-axis values for the curve, click the Setup button.
The setup dialog appears, displaying the wells used in the analysis, the Sample ID,
and Quantity.
The Sample ID is derived from the original FCS file. If a sample ID was not entered for
each well, the default well name appears (A01, A02).
4
Enter the Quantity (concentration) for each well. Quantity defines the x-axis values for
the graph. These are user-defined and must be entered for each curve.
If, during acquisition, you enter a concentration as the sample ID, you can click the
Use Sample ID button to enter the Quantity. Characters before the “space” in the ID
are used for the quantity. For example, if your sample ID is “3 um staurosporine,” the
3 is used for the quantity. You can also type the values into the Quantity fields. Enter
the actual number (.01, .001, .0001) or use scientific notation (1e-2, 1e-3, 1e-4).
IC-50/EC-50
4 - 45
5
Click OK to display the graph.
The EC-50 (IC-50) value is marked by a bold + on the curve.
Toggles between EC-50 and IC-50
what is this field for?
Select between EC-50 and IC-50.
You can make the following adjustments to the data:
• Change the target value (from 20–80). For example, instead of viewing a graph
where 50% of the effect is observed, set the EC or IC value to 25 to see where
25% of the effect is observed.
• The Conc field provides the numerical value for the EC-50 point, in this case 0.16.
• Select between EC-50 and IC-50.
Special Features
Creating a Group
A group is a user-defined set of samples from one or more FCS files (or data sets).
Grouping allows you to extract and analyze a subset of samples from the data set(s). You
can also use grouping to apply different Methods to subsets of data that are part of a
single FCS file, to get different statistics, for example.
To create a group:
1
4 - 46
Select the FCS data file in the Data pane.
The plate map displays the samples acquired in that file.
guava easyCyte™ System User Guide
2
Drag the cursor across the appropriate wells in the plate
map, right-click the selected wells, and choose Create
group from the menu.
To select non-consecutive wells, press the Ctrl key while
clicking to select wells. The order that you select and add
the wells to the group is the order that they will be in the group.
The group name appears under the original FCS file in the Data pane. To rename the
group, double-click the name and type in the new name.
To add additional samples to an existing group, select the wells and drag them to the
group in the Data pane.
 NOTE: You can combine samples from different FCS files; however, if you select the
same/overlapping wells, the plate map and plots will display only the first of the
overlapping well(s) selected (eg, the first A01 selected).
3
To remove an individual well, right-click it and select Remove Item. The deleted well
is removed from the group and appears as an empty square in the plate map. Wells
can be deleted only from groups, not from the original FCS file. To delete a group,
select it and click the Delete Group icon in the Data pane.
 NOTE: Use the Duplicate Group icon in the Data pane to make a copy of a group.
Group under the data file from which it was created.
You can also use the icons at the bottom of the Data pane to create, open, save,
duplicate, and delete a group.
Special Features
4 - 47
Data Pooling
During analysis you can combine the data from multiple samples into a single sample.
This can be useful when analyzing rare events.
To pool samples, you must first pair a data file with a method to create an analyzed group.
1
Click a well that you wish to pool data from other wells into, then drag to select the
other wells.
2
Right-click the selected wells and choose Pool Samples from the menu.
Example:
D01 is selected and cursor is dragged through D03.
Right-click and select Pool Samples. Wells D02 and
D03 now appear with 01 label.
The data combined from all selected wells appears in the plots. The wells appear in
the plate map with the label number of the first selected well.
Combined (pooled) data for D01 through D03
appears in the plots.
To see the new combined number of events for a given population, open the Current
Run Stats window by selecting Tools > Show Current Run Stats. If stats have not
been created, click the New Stat Marker icon to the right of the plot and select New
Stat for Rx. Select Count as the Metric and click OK.
3
4 - 48
To “unpool” the data, click and drag to select the wells (in any order). Right-click the
selected wells and choose Cancel Pooled Samples.
guava easyCyte™ System User Guide
Overlaying Histograms
The histogram overlay feature allows you to superimpose histograms from multiple
samples derived from one data file. By selecting a different color for each sample, you
can see the different sample data overlaid within the same plot. Each histogram plot has
its own overlay list.
1
Click the Edit Overlay List icon (last icon in the tool bar of the histogram plot).
The Overlay Setup dialog box appears with the currently displayed sample well listed
as base in the far-left column and <current> under Sample ID.
2
To add samples to the overlay list (and plot), drag a well directly from the plate map to
either the histogram plot or the Overlay Setup dialog box. To select multiple wells,
drag to select wells or use the Ctrl key to select non-consecutive wells.
The plot will display the selected sample data in
the designated colors. When you overlay plots,
any markers you had previously set, as well as the
stats, apply to the base plot only. The overlays are
derived from the same gate as the initial plot. You
can also change the parameter displayed by
clicking the x-axis label and choosing a different
parameter.
• To change the overlay order, click the number (or “base”) in the left column and
drag it to a new location in the list.
• To remove data for an individual sample, click to select the well in the list and click
Delete.
• To hide the data for an overlay from the plot, click to remove the check mark from
the Show check box.
• Click Line Style, Line Color, and Fill Color to change the appearance of the overlay.
 NOTE: You cannot overlay sample data with different plot parameters. A message will
appear informing you that some samples do not have the required parameters.
Special Features
4 - 49
Printing Results
Before printing results, use the Print Preview option to customize the information that
prints.
1
If necessary, set up the page orientation, paper size, and margins by selecting File >
Page Setup.
2
Select File > Print Preview.
The Print Preview window appears.
3
4 - 50
Select how you wish the results to print:
• Select the plots to print by clicking the check box next to the plots. The selected
plots will print in the order they are listed. If you wish to reorder the plots in the list,
click and drag a plot to a new location in the list.
• Check to includes the statistics. The stats will appear in a table at the end of the
printout.
• Select how many samples you wish to print per page.
• Select how many rows you wish to print per sample.
• To scroll through the pages, place the cursor on the page, then click and drag (the
hand) to move the page.
guava easyCyte™ System User Guide
guava InCyte™ Troubleshooting
Problem
Possible Cause
Solutions
InCyte™ will not launch;
prompts you for an unlock
code.
An unlock code was not
entered or was entered
incorrectly.
Contact EMD Millipore
Technical Support for the
unlock code.
InCyte™ starts in Analysis
mode. Acquisition mode is
not available.
Communication problem
between instrument and
laptop.
Ensure USB cable is
connected between
instrument and laptop.
Gates and/or events in plots 1. Data file or Method is
Click on the AnalyzedGroup
disappear.
selected.
in the Analysed Data pane to
2. Data was acquired in tubes. display data and gates.
3. More than 96 samples were
acquired.
Few events, as indicated in 1. Clogged flow cell.
1. Remove sample, load
Cell Count section of
bleach, click Backflush.
Sample Information control
Follow with Quick Clean.
panel.
2. Insufficient sample volume. 2. Minimum sample volume is
90 µL.
3. Cells in suspension have
3. Click Abort. Remove
settled.
sample, mix, reload, and
reacquire.
Unexpected events
appearing in plots.
Instrument settings not
optimal. Acquiring debris.
Adjust settings so debris is
below threshold.
FSC Count under Cell
Count shows events, but
the events appear in the
wrong places in plots.
1. Sample was not stained.
1. Check sample. If
necessary, restain sample
from original suspension.
2. Check buffers used to
process cells.
2. Cell lysis.
guava InCyte™ Troubleshooting
4 - 51
Problem
Possible Cause
No events, as indicated in
Particle Count section of
Sample Information control
panel.
1. Sample tube or plate not
loaded.
2. No cells in sample.
Events appear in some
plots but not in others.
Ensure correct gate is
selected for plot in question.
1. Open plot menu, point to
Apply Gates and select
gate.
2. Check gate definition to
ensure it includes the
correct regions and
operators.
Events appear off scale in
dot plots or histograms.
Gains set incorrectly, or
samples staining brightly.
Adjust gain setting so
positive populations appear
on scale. Repeat Adjust
Settings with negative
sample. Adjust
compensation settings.
1. Ensure tube is loaded and
loader assembly is up.
2. Ensure correct sample is
loaded.
3. Clogged flow cell.
3. Remove sample, load
bleach, click Backflush.
Follow with Quick Clean
using DI water.
4. Broken flow cell.
4. Remove flow cell and
inspect for damage.
Replace if necessary.
5. Sample pump not working. 5. Run Quick Clean and
watch for fluid in waste
vial.
6. Laser not operational.
6. Allow laser to warm up for
15 min. If laser is not on,
contact EMD Millipore
Technical Support.
7. Loose fitting on minstac
7. Ensure tubing connector is
tubing (under metal plate).
secure.
Cannot resolve dim positive Dirty capillary.
staining from background
signal.
4 - 52
Solutions
guava easyCyte™ System User Guide
Perform at least one cycle of
Guava® Clean.
While acquiring samples,
select Clean & Rinse instead
of Quick Clean, and if
necessary, run it frequently.
Problem
Possible Cause
Solutions
Poor resolution between
positive and negative
populations.
1. Gains too low to detect
fluorescent signals.
1. Adjust settings to increase
fluorescent signal. Adjust
compensation settings.
2. Ensure positive control is
staining adequately and
with correct reagent.
2. Incomplete staining with
fluorescent probe, or
fluorescent probe
inappropriate for cell type.
3. Fluorescent probes overexposed to light, stored
improperly, or expired.
3. Refer to reagent package
insert for proper storage
instructions. Do not expose
reagent to excessive light.
Do not use expired
reagents.
4. If using antibody-based
4. Non-specific binding of
probes, try Fc blocking
fluorescent probes.
reagent during staining to
minimize non-specific
binding. Otherwise, titer
the fluorescent probes
down to reduce the
nonspecific staining.
5.
Adjust
settings to increase
5. Background noise too high.
FSC threshold to remove
debris. Or, wash stained
sample and reacquire.
Poor resolution between
positive populations.
1. Incomplete staining with
reagent(s).
1. Check expiration date and
amount of reagent(s) used
in staining.
2. Too much reagent in
2. Washing cells may remove
staining tube.
residual reagent.
3. Fluorescence background 3. Washing cells may remove
too high.
residual reagent.
4. Gain too high causing
4. Adjust settings to reduce
signal to bleed into other
gain. Adjust compensation
parameters.
settings.
5. Gain too low to optimally
5. Adjust settings to increase
detect positive signal.
gain. Adjust compensation
settings.
6. Background noise too high. 6. Adjust settings to increase
FSC threshold to remove
debris. Or, select one of
the fluorescence
parameters as the
threshold.
guava InCyte™ Troubleshooting
4 - 53
4 - 54
guava easyCyte™ System User Guide
Appendix A
Administrator Features
Setting up Access Control
guavaSoft™ software, version 3.1, supports features which allow a laboratory
administrator in controlled environments to restrict users from access to certain features
of the guava easyCyte™ System to help ensure that standard procedures are followed
correctly. Users whose access has been restricted cannot change instrument or analysis
settings, and may only operate the guava easyCyte™ System according to previouslysaved Settings files or the instrument default settings.
Who Should Read This Section on Administrator Features
By default, after installation there are no access control restrictions, and the default “GTI”
user account has access to all features of the software. If such unrestricted access is
sufficient for your needs, then no further steps are necessary and you may begin using
the software.
You should read this section if:
• you want to use access control in your laboratory or environment, or
• you want to create new user accounts, or
• you are a network administrator and need information about the guava® system
environment in order to create guava® user accounts in a manner consistent with your
local security policy
We recommend that you contact your local network administrator for help in setting up
user accounts and access control.
Administrator Features
A-1
Access Control Levels
guavaSoft™ software, version 3.1, supports three levels of access control, Administrator,
Supervisor, and Operator. Each user account under the Windows® operating system has
one of these levels, which is assigned by the lab administrator on a case-by-case basis. A
higher access control level always has privileges of all access control levels below.
Administrator-level user accounts have unrestricted access to all features of the software
including the “Administration Configuration,” and are assumed to be guavaSoft™
software Administrators who know how to properly configure all features of the software
for supervisor-level and operator-level users.
Supervisor-level user accounts have unrestricted access to all features of the software
except the “Administration Configuration” (see page B-13) and are assumed to be expert
users who know how to properly use all features of the software at the supervisor and
operator levels.
All user accounts can:
• perform the Adjust Settings step
• retrieve instrument settings files
• start new data sets
• run the easyCyte™ System and acquire data (entering all relevant information such as
sample IDs, number of events to acquire, and dilution factor and original volume, when
applicable)
• perform Quick Clean, Backflush, and Cleaning operations
• run the easyCheck™ Procedure
• abort an acquisition, when necessary
• export data to FCS 2.0, 3.0, or CSV format
• add comments to event log
• view event log file
Operator-level user accounts cannot:
• change instrument settings (FSC gain, PM1 or PM2 voltages, pump speed)
• adjust markers or gates
• make any adjustments during the Adjust Settings step
• use Next Step, except to (a) finish Adjust Settings, or (b) terminate a normal
acquisition
• save instrument settings
A-2
guava easyCyte™ System User Guide
Before Setting Up Access Control
The guavaSoft™ software, version 3.1, installer automatically creates the three different
guava® User Groups under Windows, namely, GuavaAdmin, GuavaSupervisor, and
GuavaOperator. The correct access to the various guava® files needed to function
properly are also automatically set up by the installer.
 IMPORTANT NOTE: If you plan to connect the system to a network, be sure to contact
your local network administrator for help, since the procedures described here may
need to be modified in order to comply with security policies in effect for your local
network.
“First-Time Setup After Installation” describes steps which need to be taken only once,
after installation of the software. It explains how to set up the guava® Settings Files folder
access control.
“How to Create A New User Account” describes how to create a new user account.
“Assigning a guava® Access Control Level to a User Account” describes how to assign
the access control level (Administrator, Supervisor, or Operator) to a user account.
First-Time Setup After Installation
This section shows you after installation, the three User Groups, GuavaAdmin,
GuavaSupervisor, and GuavaOperator are automatically created. The steps to set up the
folder for the guava® Setting Files are also described in this section.
The GuavaAdmin group is used by guavaSoft™ software to allow the guava®
Administrator to configure the “Administration Configuration.” This option is available from
the guavaSoft™ software main menu to a guava® Administrator only. The GuavaAdmin
group consists of only those users who are to share responsibility for administering
Access Control. All members of the GuavaAdmin group should also be a Windows
Administrator. Only these users (or a Windows administrator) will be able to assign or
change the access control levels for other users.
Three User Groups Automatically Created Upon Installation
1
Log on as administrator. (No password is required by default.)
Administrator Features
A-3
2
Right-click My Computer and select Manage from the menu. The Computer
Management window appears. Expand the content of the “Local Users and Groups”
folder and click on the “Groups” folder.
Create the guavaSoft™ “guavaSettings” Folder
1
Double-click My Computer. Double-click Shared Documents. Create a new folder in
the Shared Documents folder called guavaSettings.
 NOTE: If using Windows® 7 OS, the Shared Documents folder does not exist under
My Computer. Request that your IT department create a guavaSettings folder that is
accessible to the appropriate users.
A-4
guava easyCyte™ System User Guide
How to Create A New User Account
1
Log on as administrator.
2
Right-click My Computer and select Manage from the menu.
3
Find Local Users and Groups and click to expand its contents. Click the Users folder.
4
Select New User from the Action menu in the menu bar at the top of the window. The
New User dialog box appears.
Administrator Features
A-5
5
Enter a user name to be added to the system. Type in a full name (first and last), a
password and confirmation, and a description. In the example below, a user account
“User1” for “Joe Smith” in “R&D” is created.
6
Click Create. You may continue adding more users by repeating the steps starting
with step 4. Click Close when you are finished adding users.
7
You will need to assign each user to the proper access control group. Proceed to
“Assigning a guava® Access Control Level to a User Account” for this information.
Assigning a guava® Access Control Level to a User Account
This section shows you how to assign the Access Control level to a user account for
guavaSoft™ software. You do this by assigning the user to one of the groups created in
“First-Time Setup After Installation” on page A-3.
If you want the user to be able to administer Access Control for other users or to be able
to configure the “Administration Configuration,” (normally, very few users should have this
right), add the user to the GuavaAdmin group as shown below. Also make sure that the
user you have added to the GuavaAdmin group is also a Windows Administrator.
In a similar manner, a user account can be assigned to have either Supervisor- or
Operator-level access control simply by adding the user to either the GuavaSupervisor or
GuavaOperator group, respectively, as shown below.
Be careful not to assign a user to more than one guava® group. The higher Group
privilege takes precedence, which may not be what you want.
A-6
1
Log on as a Windows Administrator.
2
Right-click My Computer and select Manage from the menu.
3
Navigate to the Groups folder under “Local Users and Groups.”
guava easyCyte™ System User Guide
How to Give a User Account GuavaAdmin-Level Access
1
Right-click on the GuavaAdmin group and select Properties.
2
Click Add in the GuavaAdmin Properties window.
The Select Users window appears.
3
Enter the user login name for the administrator and click OK.
4
Repeat these steps to add additional administrators.
 NOTE: If a user in the GuavaAdmin group is NOT a Windows Administrator, you can
add the user to the Administrators group:
• Right-click My Computer and select Manage from the menu.
• Click the Groups folder under the “Local Users and Groups” folder.
• Right-click on the “Administrators” group in the right side of the window and select
Add to Group from the menu.
• Click Add in the Administrators Properties window.
• Select the user you want to add to the Administrators group and click Add.
• Click OK when you are finished.
How to Give a User Account GuavaSupervisor-Level Access
1
Right-click on the GuavaSupervisor group and select Properties.
2
Click Add in the GuavaSupervisor Properties window.
The Select Users window appears.
3
Enter the user login name for the supervisor and click OK.
4
Repeat these steps to add additional supervisors.
Administrator Features
A-7
How to Give a User Account GuavaOperator-Level Access
1
Right-click on the GuavaOperator group and select Properties.
2
Click Add in the GuavaOperator Properties window.
The Select Users window appears.
3
Enter the user login name for the operator and click OK.
4
Repeat these steps to add additional operators.
5
Reboot the system when finished. Click the Start button, click Turn Off Computer,
click Restart.
Information For Network/IT Administrators
• User accounts which function as administrators for guava® Access Control do not
need to have Windows Administrator privileges, they only need full control of the
AccessRights folder within the guavaSoft™ folder hierarchy.
• The guavaSettings folder described in “First-Time Setup After Installation” does not
need to be in C: drive. It can be located anywhere as long as all users can access it.
Although the setup procedures above do not detail how to do this, the Operator-level
accounts do not need access to the guavaSettings folder, since they cannot create
Settings files. Therefore, you could obtain greater security by arranging that the
GuavaOperator Group has only Read access to the guavaSettings folder.
• All guavaSoft™ software users need Read/Write/Modify access to the Log folder when
running the easyCheck™ Procedure, since this assay updates the GuavaCheckLog.
Administration Configuration
Administrators can configure or customize certain software features for supervisors and/
or operators.
You must be logged on as an administrator to gain access to the Setup button on the
guavaSoft™ software main menu.
A-8
guava easyCyte™ System User Guide
1
Click Setup from the main menu.
Available to administrators
who log onto the system.
The Customize guavaSoft screen appears.
2
Select Configure under Access Rights.
Administrator Features
A-9
3
Click the check boxes for the features you wish to allow for supervisors and/or
operators.
The following table describes each feature.
Feature
Description
Disable Append to File
If selected, will not allow user to append to an existing FCS
3.0 file. Users may overwrite the existing file or create a
new file. This applies to a data file when you click New
Data Set (or Start New Data Session File for InCyte™) at
the start of a session.
If overwriting is disabled (see below), you will be appending
to a copy of the existing file since you cannot overwrite it. If
overwriting is allowed, you will be appending to the end of
the existing file.
Disable Overwrite File
If selected, will not allow users to overwrite an existing FCS
3.0 file. Users may append to the existing file or create a
new file. This applies to a data file when you click New
Data Set (or Start New Data Session File for InCyte™) at
the start of a session, as well as to a data file when you
make changes during analysis.
Require easyCheck Fields
If selected, the user must enter values for the Bead Lot #,
Bead Expiration Date, and Expected Particles/mL before
starting every easyCheck™ run. If this option is not
selected, the information will default to the last information
entered.
Require non-blank ViaCount
Fields
If selected, the user must enter values for the Reagent Lot
# and Reagent Expiration Date before a ViaCount Assay
can be started. If this option is not selected, the reagent
information is not required.
A - 10 guava easyCyte™ System User Guide
4
Feature
Description
Include Full User Name on
Printouts
If selected, the user’s full name is included on all printouts
generated by that user. This information is taken from the
“Full Name” field in the user’s Windows XP account record.
Include “Reviewed By Field”
on Printouts
If selected, places a Reviewed by: __________ field on all
printouts generated by the user.
Disable retrieval of InCyte
Settings
If selected, users cannot adjust instrument settings or
retrieve InCyte™ Methods, instrument settings, and
compensation settings from individual Method or
instrument settings files. Methods, instrument settings, and
compensation settings can only be retrieved from a single
FCS file.
Click OK to save the settings.
Administrator Features
A - 11
A - 12 guava easyCyte™ System User Guide
Appendix B
Glossary
acquisition
The electronic and software function of collecting various
types of information from a cell sample.
Analysed Group
An FCS file created in InCyte™ software. It contains the
sample data and the Analysis Method. When data is
acquired in InCyte™, the software will automatically pair the
data set and Method for analysis. If data were acquired in
any other guavaSoft™ module, you will need to pair the data
with a new or existing Method during analysis to form the
Analysed Group.
analysis
The software function of numerically and graphically
manipulating data to identify and separate cell populations for
the purpose of calculating relevant statistical information.
antibodies
A class of proteins secreted by sensitized B lymphocytes
following contact with an antigen. Also referred to as
immunoglobulins. Monoclonal antibodies, derived from a
unique secreting clone of the parent B cell, are characterized
by their highly specific antigen binding capabilities.
area
The area under the electronic pulse. The area measurement
provides a more accurate assessment of total signal
fluorescence.
caspases
Cysteinyl-directed aspartate-specific proteases. A family of
enzymes that initiate the apoptotic cascade, carry out cellular
breakdown, and process cytokines.
Glossary
B-1
B-2
coefficient of
variation (%CV)
The ratio of the standard deviation to the mean, expressed
as a percent. It is calculated using the formula:
%CV = SD x 100
x
compensation
A process of reducing the unwanted fluorescence signal of
one fluorochrome overlapping into the range of wavelengths
of another fluorochrome.
data set
A series of samples included within one file for a selected
assay. An FCS file and a spreadsheet file are saved for each
data set.
depolarized cells
Cells in which the mitochondrial membrane potential has
collapsed. Because of this loss of negative potential, the
JC-1 dye no longer accumulates in the mitochondria, causing
cells to fluoresce mostly green.
detector
A device used to measure light intensity. The fluorescence
detectors are photomultiplier tubes (PM1, PM2, and PM3)
and the FSC detector is a photodiode. Both output a current
that is proportional to the intensity of incident light.
dot plot
A graphical representation of two-parameter data. Each axis
of the plot displays values for one parameter. A dot
represents the values for a cell or particle.
FBS
Fetal bovine serum. A cell-free extraction of blood obtained
from fetal calves used to supplement growth media in tissue
culture.
FCS file
Flow Cytometry Standard file. A data file containing the
results for an individual sample as well as all acquisition
information at the time of data collection. FCS files are
defined by the Data Files Standards Committee of the
Society for Analytical Cytology. Cytometry. 1990;11:323–332.
flow cell
An optical assembly within the guava easyCyte™ System.
The flow cell consists of a metal shuttle holding a glass
capillary with a tiny chamber where the laser beam
illuminates the sample stream and cellular measurements
occur.
guava easyCyte™ System User Guide
fluorescence
The phenomenon of light emission that occurs when a
fluorochrome’s excited electrons drop to a lower energy level.
fluorochrome
A fluorescent dye used as a detection reagent in cell analysis
applications. A molecule capable of absorbing light at a
certain wavelength, then emitting light at a longer wavelength
(fluorescence) as it releases energy. For a list of all
fluorochromes compatible with the easyCyte™ System, see
“Fluorochromes” on page 4 of the Specifications section.
FSC
Forward scatter. Light scattered as a particle passes in front
of the laser beam. Forward scattered light is used as an
indicator of relative particle size and incorporates both the
particle’s cross-sectional dimension and refractive index.
gate
A graphical boundary that defines a subset of data. Gates
may be set on a single-parameter histogram or a twoparameter dot plot.
helper/inducer T cells
T cells that promote the immune response by releasing
soluble helper factors such as interleukin 2 and interleukin 4.
histogram
A graphical representation of 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 (cells).
isotype control
An antibody of the same immunoglobulin class and
fluorescent capacity as the test reagent, but having no
specificity for the target antigen or other antigens present on
the test cells. The isotype control is used to estimate
nonspecific binding of the test reagent.
laser
Light amplification by stimulated emission of radiation. A light
source that is highly directional, monochromatic, coherent,
and bright. The emitted light is in one or more narrow spectral
bands, and is concentrated in an intense, narrow beam.
marker
A boundary or set of boundaries used to segregate data into
subsets for statistical analysis. Set a marker on a histogram
to obtain statistics on a certain region. Set quadrant markers
on a dot plot to obtain statistics on data within four quadrants.
Glossary
B-3
B-4
mean fluorescence
The average of the fluorescence intensities of each event
acquired within a given set of events.
median
The axis value for the event that falls in the middle of the
distribution.
Method
The part of an InCyte™ FCS file that contains all the analysis
components (gates, regions, markers, plots, parameters, and
statistical setup). Methods can be part of the FCS file and can
also be saved to a separate file (.gsy). Data files acquired
using a program other than InCyte™ will not have Methods
associated with them, therefore you must use a new or
existing Method before you can analyze the file using
InCyte™.
monocytes
Immature macrophages found in the blood.
NK cells
Natural killer cells. Non-T, non-B lymphocytes found in
normal individuals and capable of killing some tumor cells
and some virus-infected cells.
offset
A baseline detector setting for an individual assay to ensure
that the cells are being detected.
parameter
A specific cell property that is measured as the cell passes in
front of the laser beam. Each parameter is the output from a
photomultiplier (which measures fluorescence) or a
photodiode (which measures forward scatter).
PMNs
Polymorphonuclear cells. Myeloid cells that are derived from
the bone marrow. These cells possess a lobulated, irregular
nucleus and cytoplasm that is filled with granulocytes. PMNs
consist of neutrophils, eosinophils, and basophils.
PMT
Photomultiplier tube. A device used for measuring light
intensity.
polarized cells
Cells which exhibit a normal mitochondrial membrane
potential, and hence when exposed to the JC-1 dye,
fluoresce mostly orange.
guava easyCyte™ System User Guide
population
A group of cells that express similar values within one or
more parameters. For example, cells that are positive for a
particular antibody appear in the same location within a
histogram or dot plot.
SR-VAD-FMK
Sulforhodamine-valyl-alanyl-aspartyl-fluoromethylketone.
A cell-permeable, noncytotoxic caspase inhibitor that
covalently binds to multiple caspases that have been
activated during apoptosis.
SSC
Side scatter. Light scattered as a particle passes in front of
the laser beam. Side scattered light is measured at
approximately 90° from the incident laser beam. Side
scattered light is used as an indicator of a particle’s relative
internal granularity.
suppressor/cytotoxic
T cells
T cells that suppress the response of other cells to antigen,
or kill other cells in an antigen-specific manner.
T cells
Lymphocytes that have undergone a period of processing in
the thymus and are responsible for mediating the cellmediated immune response.
threshold
The minimum level of discrimination to electronically
eliminate unwanted signal. A threshold setting allows you to
specify events you wish to acquire based on signal intensity
of the event. Anything below the threshold in not acquired.
width
The width of the electronic pulse. The width measurement
helps you discriminate doublets from singlets.
Glossary
B-5
B-6
guava easyCyte™ System User Guide
Appendix C
Ordering Information
For ordering information contact the nearest EMD Millipore office by calling 1 800 645-5476,
or visiting us on our website at www.millipore.com/offices.
EMD Millipore and its distribution network will provide guava® products to all sectors of
life science research in certain countries outside North America and Europe.
Instrument Components
Parts
Catalog Number
guava easyCyte™ 12 System (software not included)
0500-5012
guava easyCyte™ 8 System (software not included)
0500-5008
guava easyCyte™ 6-2L System (software not included)
0500-5007
guava easyCyte™ 5 System (software not included)
0500-5005
laptop computer
0110-3670
sample tube holder, titer tubes
0100-0060
sample tube holder, 1.5-mL microcentrifuge tubes
0100-2180
cleaning solution vial assembly (small, metal tubing connector)
0110-3030
(large, plastic tubing connector)
waste vial assembly (small, metal tubing connector)
(large, plastic tubing connector)
0110-8125
0110-3020
0110-8120
flow cell assembly
0500-2270
flow cell tightening tool
6000-2410
flow cell removal tool (tweezer model)
6000-3020
syringe assembly cleaning tool
0110-0210
guava easyCyte™ System User Guide text
0110-8493
instrument shipping box
0110-2640
Software
guava easyCyte™ System Software Modules
Catalog Number
guava® ExpressPro Software Module (required for 6-color operation)
0500-4125
guava InCyte™ Software Module
0500-4120
guavaSuite Software Modules for the guava easyCyte 12 System
0500-4130
Ordering Information
C-1
Essential Tools
Kit
guava easyCheck™ Kit (50 tests)
Guava® ICF (Instrument Cleaning Fluid) [100 mL]
Catalog Number
4500-0025
4200-0140
Reagent Kits
Cell Health
ViaCount Assay Kits
Guava® ViaCount Reagent (100 tests)
Guava® ViaCount Reagent (600 tests)
Guava® ViaCount Flex Reagent (100 tests)
Guava® ViaCount Flex Reagent (500 tests)
CDR (Cell Dispersal Reagent) [100 tests]
Cell Cycle Kits
FlowCellect® Bivariate Cell Cycle Kit for DNA Replication Analysis (25 tests)
FlowCellect® Bivariate Cell Cycle Kit for G2/M Analysis (25 tests)
Guava® Cell Cycle Reagent (100 tests)
DNA Damage Kits
FlowCellect® Multi-Color DNA Damage Response Kit (25 tests)
FlowCellect® DNA Damage Histone H2A.X Dual Detection Kit (25 tests)
FlowCellect® Cell Cycle Checkpoint H2A.X DNA Damage Kit (25 tests)
FlowCellect® Cell Cycle Checkpoint ATM DNA Damage Kit (25 tests)
FlowCellect® Histone H2A.X Phosphorylation Assay Kit
MitoHealth Kits
FlowCellect® MitoPotential Red Kit (100 tests)
FlowCellect® MitoDamage Kit (100 tests)
FlowCellect® MitoLive Kit (100 tests)
FlowCellect® MitoStress Kit (100 tests)
FlowCellect® Cytochrome c Kit (100 tests)
FlowCellect® Oxidative Stress Characterization Kit (25 tests)
guava® MitoPotential Kit (100 tests)
Apoptosis Kits
Early Apoptosis Kits
FlowCellect® Annexin Red Kit (100 tests)
guava® Nexin Reagent (100 tests)
guava® Nexin Reagent (500 tests)
Mid Apoptosis Kits
guava® MultiCaspase SR Kit (100 tests)
guava® Caspase 9 SR Kit (100 tests)
guava® MultiCaspase FAM Kit (100 tests)
guava® Caspase 3/7 FAM Kit (100 tests)
guava® Caspase 8 FAM Kit (100 tests)
guava® Caspase 9 FAM Kit (100 tests)
guava® MultiCaspase SR and Caspase 3/7 FAM Kit (100 tests)
guava® MultiCaspase SR and Caspase 8 FAM Kit (100 tests)
C-2
guava easyCyte 12 System User Guide
Catalog Number
4000-0040
4000-0041
4500-0110
4700-0060
4700-0050
FCCH025102
FCCH025103
4500-0220
FCCH025104
FCCS025153
FCCH025142
FCCH025143
FCCS100182
FCCH100105
FCCH100106
FCCH100107
FCCH100109
FCCH100110
FCCH025111
4500-0250
FCCH100108
4500-0450
4500-0455
4500-0500
4500-0520
4500-0530
4500-0540
4500-0550
4500-0560
4500-0570
4500-0580
Cell Health
guava® MultiCaspase SR and Caspase 9 FAM Kit (100 tests)
guava® Caspase 9 SR and Caspase 3/7 FAM Kit (100 tests)
guava® Caspase 9 SR and Caspase 8 FAM Kit (100 tests)
guava® Caspase 9 SR and MultiCaspase FAM Kit (100 tests)
Late Apoptosis Kits
guava® TUNEL Kit (100 tests)
Apoptosis Signaling Kits
FlowCellect® Bcl-2 Activation Dual Detection Kit (25 tests)
Autophagy
FlowCellect® GFP-LC3 Reporter Autophagy Assay Kit (CHO) [25 tests]
FlowCellect® Autophagy Reagent Pack (25 tests)
FlowCellect® GFP-LC3 Reporter Autophagy Assay Kit (U20S) [25 tests]
FlowCellect® RFP-LC3 Reporter Autophagy Assay Kit (25 tests)
FlowCellect® Autophagy LC3 Antibody-based Assay Kit (100 tests)
Catalog Number
4500-0590
4500-0630
4500-0640
4500-0650
Immunology
Intracellular Cytokine Kits
Helper T Cell Kits – Mouse
FlowCellect® Mouse Th1 Intracellular Cytokine Kit (25 tests)
FlowCellect® Mouse Th2 Intracellular Cytokine Kit (25 tests)
FlowCellect® Mouse Th17 Intracellular Cytokine Kit (25 tests)
FlowCellect® Mouse Th1/Th2 Intracellular Cytokine Kit (25 tests)
FlowCellect® Mouse Th1/Th17 Intracellular Cytokine Kit (25 tests)
FlowCellect® Mouse Th1 Differentiation Tool Kit (25 tests)
FlowCellect® Mouse Th2 Differentiation Tool Kit (25 tests)
FlowCellect® Mouse Th17 Differentiation Tool Kit (25 tests)
FlowCellect® Mouse Treg Differentiation Tool Kit (25 tests)
T-Cell Kits – Human
FlowCellect® Human FOXP3 Treg Characterization Kit (25 tests)
FlowCellect® Human CD4/CD8 T Cell Kit (100 tests)
T-Cell Kits – Mouse
FlowCellect® Mouse FOXP3 Treg Identification Kit (25 tests)
FlowCellect® Mouse Viable Treg Characterization Kit (25 tests)
B-Cell Identification Kits – Human
FlowCellect® Human Memory B Cell Identification Kit (25 tests)
FlowCellect® Human B Cell FAS Kit (100 tests)
B-Cell Identification Kits – Mouse
FlowCellect® Mouse Breg Identification Kit (25 tests)
Immune Cell Health - Human
FlowCellect® Human T Cell Apoptosis Kit (100 tests)
FlowCellect® T Cell MitoDamage Kit (100 tests)
FlowCellect® T Cell Activation Kit (100 tests)
FlowCellect® Human CD8 T Cell FAS Kit (100 tests)
FlowCellect® Human CD4 T Cell FAS Kit (100 tests)
FlowCellect® Human T Cell Caspase 3/7 Kit (100 tests)
Catalog Number
4500-0121
FCCS025108
FCCH100170
FCCF200097
FCCH100181
FCCH100183
FCCH100171
FCIM025123
FCIM025124
FCIM025125
FCIM025137
FCIM025138
FCIM025161
FCIM025162
FCIM025163
FCIM025166
FCIM025118
FCIM100158
FCIM025126
FCIM025168
FCIM025159
FCCH100137
FCIM025154
FCCH100138
FCCH100139
FCCH100141
FCCH100140
FCCH100154
FCCH100157
Ordering Information
C-3
Immunology
FlowCellect® Human T Cell Caspase 8 Kit (100 tests)
FlowCellect® Human T Cell Caspase 9 Kit (100 tests)
guava® CellToxicity Kit (100 tests)
guava® CellGrowth Kit (200 tests)
Catalog Number
FCCH100155
FCCH100156
4500-0230
4500-0270
Cell Signalling
MAPK Pathway
FlowCellect® PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit (25 tests)
FlowCellect® EGFR/MAPK Pathway Activation Detection Kit (25 tests)
FlowCellect® MAPK Activation Dual Detection Kit (25 tests)
FlowCellect® p38 Stress Pathway Activation Detection Kit (25 tests)
EGFR Pathway
FlowCellect® EGFR/MAPK Pathway Activation Detection Kit (25 tests)
FlowCellect® EGFR/RTK Activation Dual Detection Kit (25 tests)
PI3/Akt/m-TOR Pathway
Catalog Number
FlowCellect® PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection Kit (25 tests)
FlowCellect® PI3K Activation Dual Detection Kit (25 tests)
FlowCellect® PI3K-mTOR Signaling Cascade Kit (25 tests)
Jak/STAT Pathway
FlowCellect® EGFR/STAT3 Pathway Activation Detection Kit (25 tests)
FlowCellect® Multi-STAT Activation Profiling Kit (25 tests)
FlowCellect® STAT1 Activation Dual Detection Kit (25 tests)
FlowCellect® STAT3 Activation Dual Detection Kit (25 tests)
Multiple Pathway
FlowCellect® PLC-gamma 1 Activation Dual Detection Kit (25 tests)
FlowCellect® Src Activation Dual Detection Kit (25 tests)
Apoptosis Signaling Pathway
FlowCellect® Bcl-2 Activation Dual Detection Kit (25 tests)
Chemokine Receptor Kits
FlowCellect® Chemokine Receptor CCR1 Surface Expression Identification and Quantification
Kit (25 tests)
FlowCellect® Chemokine Receptor CXCR6 Surface Expression Identification and Quantification
Kit (25 tests)
Stem Cell Kits
Human
FlowCellect® Human NK Cell Characterization Kit (25 tests)
Rodent
FlowCellect® Rodent NSC Characterization Kit (Neural) [25 tests]
C-4
guava easyCyte 12 System User Guide
FCCS025100
FCCS025101
FCCS025106
FCCS025132
FCCS025101
FCCS025107
FCCS025100
FCCS025105
FCCS025210
FCCS025111
FCCS025550
FCCS025142
FCCS025143
FCCS025145
FCCS025154
FCCS025108
FCCR100410
FCXR600424
FCIM025164
FCRNC25112
Service Plans
Service Total Plans
Catalog Number
guava easyCyte™ 5 Service Total 1-yr plan (at time of purchase)
0500-5300
guava easyCyte™ 5 Service Total 1-yr plan (after purchase)
0500-5305
guava easyCyte™ 5 Service Total 2-yr plan
0500-5310
guava easyCyte™ 6-2L Service Total 1-yr plan (at time of purchase)
0500-5330
guava easyCyte™ 6-2L Service Total 1-yr plan (after purchase)
0500-5335
guava easyCyte™ 6-2L Service Total 2-yr plan
0500-5340
guava easyCyte™ 8 Service Total 1-yr plan (at time of purchase)
0500-5870
guava easyCyte™ 8 Service Total 1-yr plan (after purchase)
0500-5875
guava easyCyte™ 8 Service Total 2-yr plan
0500-5880
guava easyCyte™ 12 Service Total 1-yr plan (at time of purchase)
0500-6870
guava easyCyte™ 12 Service Total 1-yr plan (after purchase)
0500-6875
guava easyCyte™ 12 Service Total 2-yr plan
0500-6880
Preventative Maintenance Plans
Catalog Number
guava easyCyte™ 5 Preventative Maintenance 1-yr plan (at time of purchase)
0500-5285
guava easyCyte™ 5 Preventative Maintenance 1-yr plan (after purchase)
0500-5290
guava easyCyte™ 5 Preventative Maintenance 2-yr Plan
0500-5295
guava easyCyte™ 6-2L Preventative Maintenance 1-yr plan (at time of purchase)
0500-5315
guava easyCyte™ 6-2L Preventative Maintenance 1-yr plan (after purchase)
0500-5320
guava easyCyte™ 6-2L Preventative Maintenance 2-yr Plan
0500-5325
guava easyCyte™ 8 Preventative Maintenance 1-yr plan (at time of purchase)
0500-5270
guava easyCyte™ 8 Preventative Maintenance 1-yr plan (after purchase)
0500-5275
guava easyCyte™ 8 Preventative Maintenance 2-yr Plan
0500-5280
guava easyCyte™ 12 Preventative Maintenance 1-yr plan (at time of purchase)
0500-6370
guava easyCyte™ 12 Preventative Maintenance 1-yr plan (after purchase)
0500-6375
guava easyCyte™ 12 Preventative Maintenance 2-yr Plan
0500-6380
On-Site Instrument Training and Application Support
Part Number
guava easyCyte™ Installation and Basic Training (half day)
0500-1690
guava easyCyte™ Installation and Advanced Training (full day)
0500-1680
Installation Qualification/Operation Qualification (IQ/OQ)
Part Number
guava easyCyte™ IQ/OQ (customer performed)
8000-1995
guava easyCyte™ IQ/OQ (EMD Millipore performed)
8000-1996
Ordering Information
C-5
Additional Supplies
The following materials can be ordered directly from the manufacturer.
C-6
Tubes
Catalog Number
Microcentrifuge tubes, 1.5-mL, conical bottom, screw cap
• VWR International–clear tubes
• VWR International–screw caps
• VWR International–tubes with caps
• Fisher Scientific–clear tubes
16466-030
89004-346
60872-324
02-681-339
Microtiter tubes, 1.2-mL conical bottom, no caps
• E & K Scientific–tubes
• VWR International–tubes
604508-RC
46300-494
guava easyCyte 12 System User Guide
Specifications
guava easyCyte™ System
Operating Environment
temperature:
humidity:
power:
fuse rating:
Instrument size:
height:
width:
depth:
Instrument weight:
laptop size:
height:
width:
depth:
laptop weight:
Optics
lasers:
forward scatter detector:
side scatter detector:
fluorescence detectors:
Signal Processing
parameter dynamic range:
pulse processing:
time:
16°–30°C (60°–86°F)
10–90% relative humidity (non-condensing)
100–240 VAC, 50/60 Hz, 120W
USA and Japan (110 V): 2.5 A, 250 V Time-lag
Europe: (220-240 V): 1.6 A, 250 V Time-lag (x2)
9.1 in (23.1 cm)
17.7 in (45 cm)
17.5 in (44.4 cm)
35 lb (15.9 kg)
11.5 in (29.2 cm) when open
14.2 in (36.1 cm)
10.4 in (26.4 cm)
8 lb (3.6 kg)
violet laser (405 nm), 100 mW
blue laser (488 nm), 150 mW
red laser (642 nm), 100 mW
photodiode
photodiode
See “easyCyte Lasers and Fluorescent Filters” on the
following page.
3.5 decades (except as noted below):
4.0 decades (guava® ExpressPro, and Caspase software
module)
5.0 decades (guava InCyte™ software module)
digital signal processing
every particle time stamped
Specifications
1
Performance
counting accuracy:
counting precision:
Fluidics
flow cell dimension:
pump:
sample flow rate:
cleaning and waste vials:
waste generation:
dead volume:
sample concentration:
sample requirement:
Data Management
computer:
data file structure:
2
±10%
≤10% CV
standard square capillary with ID of 100 µm
positive displacement
7 µL/min to 36 µL/min (7 µL/min to 72 µL/min for guava
InCyte™, ExpressPro, and Caspase modules)
15-mL glass vials with screw tops
typically <40 mL in 8 hours of continuous use
50 µL (for 1.5-mL tubes)
final particle concentration of 104 to 106 particles/mL for
accurate results
as few as 2,000 cells/test; typically 25,000–100,000 cells/
test depending on the assay
Dell™ laptop running Windows 7 Ultimate (32-bit only), and
including Microsoft Excel. Minimum configuration: Intel
Core i5-3210M processor (2.5 GHz, 3M cache); 4 GB,
DDDR3-1600 MHz SDRAM; 320 GB hard drive; 2 USB
ports
Output data file formats:
• binary data storage in Flow Cytometry Standard (FCS)
3.0 format
• spreadsheet results file in comma-separated value
(CSV) format
• optional export of binary data in FCS 2.0 or 3.0 format
• optional CSV list-mode data
guava easyCyte™ System User Guide
easyCyte Lasers and Fluorescent Filters
The following table lists the laser and power for each of the easyCyte™ instruments, and
the filters used for each parameter.
Filter
Instrument
5 & 5HT
6-2L & 6-2LHT
8 & 8HT
12 & 12HT
Laser
488 nm (blue)
488 nm (blue)
488 nm (blue)
488 nm (blue)
Power
50 mW
50 mW
150 mW
150 mW
Legacy
name
New
Name
FSC
N/A
X
X
X
X
FSC
FSC
SSC
488/16
X
X
X
X
SSC
SSC
Green-B
525/30
X
X
X
X
GRN
GRN-B
Yellow-B
583/26
X
X
X
X
YLW
YEL-B
Red-B
695/50
X
X
X
X
RED
RED-B
NIR-B
785/70
N/A
N/A
X
X
NIR
NIR-B
Laser
405 nm (violet)
Power
100 mW
Blue-V
450/45
Green-V
525/30
Yellow-V
583/26
X
Red-V
695/50
X
N/A
Laser
Power
Red-R
662/15
NIR-R
785/70
N/A
N/A
N/A
X
X
BLU-V
N/A
GRN-V
YEL-V
RED-V
642 nm (Red)
642 nm (Red)
642 nm (Red)
100 mW
100 mW
100 mW
X
X
X
RED2
RED-R
N/A
X
X
NIR2
NIR-R
Specifications
3
Fluorochromes
The following tables, list all fluorochromes that can be used with the easyCyte™ System,
the laser that excites the dye, and the parameter/detector that detects the emitted signal.
Blue (448/50 nm) Green (525/30 nm) Yellow (583/26 nm)
Red (661/15 nm)
Red (695/50 nm)
NIR (785/70 nm)
DAPI
AlexaFluor™ 430
Pacific Orange
Alexa Fluor® 647
eFluor 650
PE-Alexa Fluor® 750
Hoescht 33258
Pacific Green
Brilliant Violet
APC
Brilliant Violet
Propidium Iodide
Alexa Fluor® 405
Brilliant Violet™
Odot 565
CD647
Odot 705
PE-Cy™7
Marnia Blue
Odot 525
Odot 585
Cy™5
7-AAD
APC-Cy™7
Pacific Blue™
Odot 545
Alexa 555
Odot 655
Propidium Iodide
APC-Alexa Fluor® 750
Live/Dead Violet
FITC
Alexa 568
DRAQ5
PE-Alexa Fluor® 647
DyLight 405
GFP
CF™555
Ethidium bromide
PE-Alexa Fluor® 700
eFluor 450
AlexaFluor™ 488
CD568
Ethidium homodimer
PE-Cy™5
Zombie Aqua™
Brilliant Violet™
CF™488
®
PE-B, PE-R
®
SYTOX Red
PE-Cy™5.5
Cy™2
PE-Texas Red
TO-PRO™-3
PE-Texas Red®
FAM
Odot 565
TOTO-3
PerCP
Rhodamine 110
Acridine Orange
DilC1(5)
PerCP-Cy™5.5
Odot 525
dsRED
MitoSense Red
Odot 705
Acridine Orange
Ethidium bromide
BODIPY™ 650/665
DRAQ5
EGFP
Ethidium homodimer
Ethidium bromide
EYFP
SYBR Gold
Ethidium homodimer
Thiazole Orange
SYBR Green
LDS-751
®
SYBR Gold
SYTOX Orange
SYBR Green
TO-PRO™-1
SYTO® BC
JC-1
SYTOX®
TMRE
Green
YO-PRO™-1
TRMR
YO-YO™-1
CFSE
DiOC6(3)
Nile Red
Nile Red
JC-1
Rhodamine 123
BODIPY™-FL
Calcien
CFSE
Oregon Green™
4
guava easyCyte™ System User Guide
405-nm laser
488-nm laser
642-nm laser
Compliance
The guava easyCyte™ System contains three lasers:
• a Class IIIb laser operating at 405 nm in CW mode
• a Class IIIb laser operating at 488 nm in CW mode
• a Class IIIb laser operating at 642 nm in CW mode
Light shields within the instrument enclose the path of laser radiation. Additionally, the
instrument enclosure provides secondary protection from any laser radiation.
This product complies with:
• CFR (Code of Federal Regulations) Chapter 1, Subchapter J and installation
(overvoltage) category II
• Class 1 limits for exposure to laser radiation set by the Center for Devices and
Radiologic Health (CDRH)
• EN 60825-1:2007, EN 61010-1:2010, and EN 61326-1:2013
• CAN/CSA-C22.2 No. 61010-1-04, UL Std. No 61010-1 (2nd Edition)
Symbols
Attention, consult accompanying documents.
Affixed in accordance with European Council Directive 73/23/EEC
Danger, laser radiation
C
US
In accordance with Canadian Standards Association
Separate collection of waste at end of life as required by European Directives.
Dispose of in accordance with the applicable country regulation.
Dangerous voltage
Power on
Power off
Specifications
5
Notice to Purchaser: Limited Use License
Use of this product is covered by one or more of the following US patents and
corresponding patent claims outside the US: (5,798,222; 6,403,378; 6,710,871;
6,816,257; 7,320,775; 7,410,809; 8,184,271 and 7,847,923). The purchase of this
product includes a limited, non-transferable immunity from suit under the foregoing patent
claims for using this product by the purchaser for its own internal research. No right under
any other patent claim, no right to perform any patented method, and no right to perform
commercial services of any kind, including, without limitation, reporting the results of
purchaser's activities with use of the product for a fee or other commercial consideration,
is conveyed by express implication, or by estoppel. This product is intended for research
use only. Diagnostic uses require a separate use license from EMD Millipore. Further
information on commercial licenses may be obtained by contacting the Director of
Licensing, as listed on EMD Millipore's website:
http://www.emdmillipore.com/proprietary-technologies-available-for-licensing/
c_bhCsHfETIxYAAAE1gKU2Vc2d?back=true
The Product has not been tested by EMD Millipore for safety and efficacy in food, drug,
device, cosmetic, commercial or any other use.
The Product is not a medical device for the purposes of the Medical Devices Regulations
2002 or any other Regulations derived from Directive 98/79/EC (“Medical Devices”), and
the Product is not in any circumstances to be sold or distributed for use as a Medical
Device.
EMD MILLIPORE AND ITS AFFILIATES shall not, no matter how such liability may be
purported to arise, except in the case of personal injury or death arising directly out of the
gross negligence, recklessness or willful misconduct of EMD MILLIPORE OR ITS
AFFILIATES, be liable to the Purchaser, or purchaser's affiliates or purchaser's
customers, employees or students, for any loss or damage (including consequential
damage or economic loss or loss of profits) arising out of, or in connection with, the sale
or use of the Product.
TO THE FULLEST EXTENT PERMITTED BY APPLICABLE LAW, IN NO EVENT SHALL
EMD MILLIPORE'S LIABILITY ARISING OUT OF THE MANUFACTURE, SALE,
SUPPLY, USE OR DISPOSITION OF THE PRODUCT, WHETHER BASED ON
WARRANTY, CONTRACT, TORT OR OTHERWISE, EXCEED THE ACTUAL PRICE
PAID BY PURCHASER FOR THE PURCHASED PRODUCT.
6
guava easyCyte™ System User Guide
Warranty
EMD Millipore Corporation (“Millipore”) warrants its products will meet their applicable
published specifications when used in accordance with their applicable instructions for a
period of one year from shipment of the products. MILLIPORE MAKES NO OTHER
WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF
MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty
provided herein and the data, specifications, and descriptions of Millipore products
appearing in Millipore's published catalogues and product literature may not be altered
except by express written agreement signed by an officer of Millipore. Representations, oral
or written, which are inconsistent with this warranty or such publications are not authorized,
and if given, should not be relied upon.
In the event of a breach of the foregoing warranty, Millipore’s sole obligation shall be to
repair or replace, at its option, the applicable product or part thereof, provided the customer
notifies Millipore promptly of any such breach. If after exercising reasonable efforts,
Millipore is unable to repair or replace the product or part, then Millipore shall refund to the
Company all monies paid for such applicable Product. MILLIPORE SHALL NOT BE
LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL, OR ANY OTHER DAMAGES
RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY
COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS.
© 2014 EMD Millipore Corporation. All rights reserved. No part of these works may be
reproduced in any form without permission in writing.
Unless otherwise stated in our catalog or other company documentation accompanying the
product(s), our products are intended for research use only and are not to be used for any
other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro
diagnostic uses, ex vivo or in vivo therapeutic uses, or any type of consumption or
application to humans or animals.
Specifications
7
8
guava easyCyte™ System User Guide
Index
A
Abort button 1-16
Acquire Next Sample button 1-15
Acquisition screen 1-14 to 1-17
adjusting settings
InCyte assay 4-8 to 4-11
analysis
InCyte 4-13 to 4-32
Analysis screen 1-18 to 1-19
area, pulse measurement
InCyte 4-9
assays
Guava InCyte 4-6 to 4-19
Autostart Acquisition feature 1-16
B
Backflush button 1-16
backflushing fluid system 1-7, 3-4
backgating 4-24
beads, easyCheck 2-3
bleach solution
adding to waste vial 3-6
for backflushing 3-4
for cleaning 3-3
for Quick Clean 3-1
buttons
Abort 1-16
Acquire Next Sample 1-15
acquisition 1-15
assay 1-9
Backflush 1-16
Cleaning 1-9
easyCheck 1-9
Exit 1-10
Next Step 1-15
Quick Clean 1-16
Save and Close Current Sample 1-15
Setup 1-9
Start Worklist 1-15
stop and close session 1-16
Stop Worklist 1-15
C
cleaning
flow cell 3-6 to 3-9
Guava Clean 3-2 to 3-3
outside of instrument 3-1
Quick Clean 3-1
solution 3-1, 3-3
cleaning solution vial 1-7
filling 3-4
clog 1-7, 3-4
compensation
during adjust settings 4-10
post acquisition 4-35
semi-automated 4-36
computer, internal
connecting to network 1-3
contour plots
InCyte 4-3
counting
beads, easyCheck 2-4
gate, InCyte 1-17, 4-11
D
data analysis
InCyte 4-13 to 4-32
deionized water
for Quick Clean 3-1
system shutdown 2-2, 3-3
dilution factor
Guava InCyte 1-17
E
easyCheck 2-3 to 2-4
beads 2-3
event log 2-6
exporting results 2-6
logging comments 2-6
reviewing previous results 2-5
trend graph 2-6, 2-7
troubleshooting 2-8
events to acquire
InCyte assay 1-17
exporting
easyCheck results to spreadsheet file 2-6
event log 1-22
InCyte results to spreadsheet file 4-33
F
FCS 2.0 data file 4-33
FCS 3.0 data file 1-20
Index
9
file name
data set 4-7
instrument settings 1-20
files
appending 1-21
FCS 2.0 4-33
FCS 3.0 1-20
overwriting 1-21
settings 1-20, 1-22
flash drive 1-7
flow cell
backflushing 3-4
cleaning 3-6 to 3-9
replacing 3-10 to 3-11
flow cytometry standard (FCS) file
exporting 4-33
fluid system 1-7
fuses, replacing 3-12
G
gate
InCyte 4-22 to 4-25
Guava Clean 3-2 to 3-3
Guava EasyCyte 1-1, 1-6 to 1-7
cleaning 3-2 to 3-3
cleaning solution vial 3-4
flow cell 3-6, 3-10
fluid system 1-7
laser 1-7
loading samples 1-6
power conditioner 1-2
waste vial 1-7, 3-5
Guava ICF 3-1, 3-3
Guava InCyte
acquisition control panel 1-17
analysis methods 4-14
assay 4-6 to 4-19
changing parameter names 4-5
compensation 4-10
contour plots 4-3
creating groups 4-46
EC50 4-44
exporting results to spreadsheet file 4-33
gating 4-22 to 4-25
HeatMap 4-38
overlaying histograms 4-49
post-acquisition compensation 4-35
printing current run stats 4-28
printing group stats 4-32
pulse area 4-9
pulse width 4-9
regions 4-19 to 4-22
setting statistical thresholds 4-43
10
guava easyCyte™ System User Guide
statistics 4-26 to 4-32
troubleshooting 4-51
unlocking 1-10
workspace 4-5
guavaSoft software 1-1
Acquisition screen 1-14 to 1-17
Analysis screen 1-18 to 1-19
main menu 1-8
overview 1-8 to 1-11
quitting 2-2
starting 2-1
H
HeatMap 4-38
I
InCyte assay 4-6 to 4-19
installing the system 1-2 to 1-3
instrument settings
adjusting 1-15
files 1-22
retrieving 1-15, 1-23
saving 1-16, 1-23
See also adjusting settings
L
laser 1-7
loading a sample 1-6
loading samples 4-8
M
minstac tubing 3-6
N
Next Step button 1-15
O
order information K-1 to K-5
original volume
Guava InCyte 1-17
overlaying histograms 4-49
P
power conditioner 1-2
printer 1-3
printing
easyCheck results 2-6
printing results
InCyte 4-28, 4-32
Q
quadrant markers
InCyte 4-27, 4-27
Quick Clean 3-1
Quick Clean button 1-16
quitting, guavaSoft 2-2
W
waste vial 1-7
emptying 3-5
width, pulse measurement 4-9
R
replacing
flow cell 3-10 to 3-11
fuses 3-12
retrieving instrument settings 1-23
running
easyCheck 2-3 to 2-4
Guava Clean 3-2 to 3-3
Quick Clean 3-1
S
sample ID
InCyte assay 1-17
when using Autostart Acquisition 1-16
sample loader
changing tube holders 1-3
loading a sample 1-6
sample number
for appended files 1-21
InCyte assay 1-17
Save and Close Current Sample button 1-15
saving instrument settings 1-23
settings files 1-20, 1-22
shutting down system 2-2
Start Worklist button 1-15
starting
guavaSoft software 2-1
system 2-1
Stop Worklist button 1-15
system
components 1-2
shutdown 2-2
startup 2-1
T
threshold setting
InCyte assay 4-10
troubleshooting
easyCheck 2-8
InCyte 4-51
tubes supported 1-3
tubing
minstac 3-6
U
unlocking InCyte 1-10
Index
11
For ordering information or technical support,
call toll-free in the USA and Canada,
Phone: +1 (800) 645-5476
Fax: +1 (951) 676-9209
For additional contact information, visit www.millipore.com
25801 Industrial Blvd.
Hayward, CA 94545-2991 USA
© 2014 EMD Millipore Corporation
All rights reserved.
Printed in the U.S.A.
0110-8493 Rev B
12/14