BIO RAD ZE5 User manual

ZE5™Cell Analyzer and
Everest™Software
User Guide
Version 2.0

ZE5 Cell Analyzer and
Everest Software
User Guide
Software Version 2.0

Bio-Rad Technical Support Department
The Bio-Rad Technical Support department in the U.S. is open Monday through Friday, 5:00 AM to
5:00PM, Pacific time.
Phone: [phone], option 2
Email: [e-mail] (U.S./Canada Only)
For technical assistance outside the U.S. and Canada, contact your local technical support office or
click the Contact us link at www.bio-rad.com.
Legal Notices
No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopy, recording, or any information storage or retrieval
system, without permission in writing from Bio-Rad.
Bio-Rad reserves the right to modify its products and services at any time. This guide is subject to
change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for
errors or omissions, or for any damage resulting from the application or use of this information.
Bio-Rad is a trademark of Bio-Rad Laboratories, Inc. in certain jurisdictions.
All trademarks used herein are the property of their respective owner.
Copyright © 2018 by Bio-Rad Laboratories, Inc. All rights reserved.

Table of Contents
Safety and Regulatory Compliance 13
Safety Warning Labels 13
Safe Use Specifications and Compliance 15
Laser Product Hazard Classification 15
Electrical Safety Information and Classification 15
Regulatory Compliance 16
Hazards 16
Biohazards 17
Chemical Hazards 17
Explosive or Flammability Hazards 18
Electrical Hazards 18
Transport 18
Operating Conditions 18
Disposal 18
Warranty 18
Chapter 1 Introduction 19
System Components 20
Installation Requirements 20
Upgrading Everest Software and ZE5 Firmware 21
Upgrading Everest Software 21
Updating ZE5 Instrument Firmware 22
Administrator and User Rights 22
Other Documentation and Training Materials 24
Chapter 2 Hardware Description 25
System Overview 25
Power and Communication Connections 26
Fluidics System 27
Sheath Bottles 28
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Table of Contents
Sheath Additive Bottle 28
System Cleaner Bottle 29
Fluidics Filters 29
Waste Bottles 30
Fluidics Connections 30
Sample Loader 31
Flow Cell 33
Optics 35
Lasers 35
Beam-Shaping Optics 36
Interrogation 36
Light Collection 36
Forward Scatter 37
Side Scatter 37
Optical Filters and Mirrors 38
The ZE5-EYE 45
Optical Filter Access Door 46
Photomultiplier Tubes 46
Electronics 47
Pre-Amplifiers 47
Analog-to-Digital Converters 47
Chapter 3 ZE5 Loader 49
Loader Components 50
Media Types 51
Probe Cleaning 53
Loader Movement 53
Chapter 4 Everest Software 55
Login Window 56
Home Window 57
Experiment Buttons 58
Help and Information Menu 59
Home Window Tools 60
Resetting Your Password 61
Recent Experiment Sessions 62
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Table of Contents
Quick Actions 63
Experiment Builder 64
Name Screen 64
Fluorophores Screen 65
Samples Screen 69
Settings Screen 83
Acquisition Workspace 89
Instrument Control Panel 89
The Toolbar 93
Details 93
Tools 93
Batch Tools 95
Quick Action and System Action Tools 95
System Tools 96
Other Toolbar Buttons 98
Status Bar 99
Notifications and System Logs 100
Chapter 5 Configuring the System 103
Managing Users 103
Creating a New User 103
Editing User Information 108
Managing User Accounts 110
Setting Global Preferences 110
Setting Plot Display Defaults and Stopping Acquisition 112
Editing QC Criteria 114
Specifying Logged Out Settings 114
Setting File Save Parameters 115
Specifying UI Preferences 116
Setting Up Vacation Mode 116
Specifying Statistics Preferences 119
Working with Optical Filter Configurations 120
Standard Filter Combinations 126
Replacing Optical Filters 130
Editing the Optical Filter Configuration 132
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Exporting Optical Filter Configurations 134
Reverting to the Default Optical Figure Configuration 134
Using the ZE5-EYE to Confirm Filter Choices 135
Installing a Neutral Density Filter 139
Chapter 6 Daily Routine 141
System Power 141
Starting Everest Software 142
Checking Fluidics Status 142
Refilling Bulk Fluidics 143
Emptying Waste Bottles 145
Replacing Sheath Bottles 146
Replacing Sheath Additive and Cleaner Bottles 148
Logging In 151
Starting Up the System 152
Running Quality Control 156
Accessing the Loader 159
Running Experimental Samples 160
Pausing the System 160
Shutting Down 161
Scheduling Automatic Startups 165
Exiting Everest Software 166
Chapter 7 Creating Experiments and Workspaces 167
Creating a New Experiment 167
Selecting Fluorophores 168
Setting Up the Run List 170
Selecting the Media Type 170
Creating a Custom Media Type 173
Plate Settings Controls 179
Setting the Run List Order 180
Activating Temperature Control 180
Specifying Shutdown Upon Completion 181
Setting Up Compensation Controls 182
Labeling Positions Manually 183
Labeling Positions Automatically 184
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Importing Names from a Spreadsheet 187
Setting Up Keywords 190
Position Settings Controls 193
Assigning Position Types 197
Selecting Standard or High-Throughput Acquisition 199
Pausing after a Tube or Well 200
Setting Volumetric Counting on Samples 200
Returning Sample to a Tube or Well 201
Setting Stop Conditions 202
Configuring Wash Settings 202
Activating Agitation 203
Specifying Flow Rate or Event Rate 203
Adding Experimental Reagents 204
Reviewing the Run List 207
Creating Plots and Histograms 209
Plots Created by the Compensation Template 209
Creating Density Plots 211
Creating Histograms 212
Creating Time Plots 213
Creating Histograms for All Channels 214
Using Plot and Histogram Tools 217
Modifying Plot Parameters 219
Managing Plot Statistics 221
Viewing and Rearranging Plot Statistics 222
Comparing Statistics 223
Adding Regions to Density Plots and Time Plots 225
Adding Bar Regions to Histograms 234
Renaming Regions 235
Applying Filters (Gates) 236
Applying Filters (Gates) Using Multiple Regions 238
Applying Gate Limits 241
Assigning Data Track Regions 242
Applying Heat Maps 244
Configuring Hit Detection 247
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Adding Diagonal Separators to Plots 249
Applying a Region to All Plots 250
Adding Annotations to Plots 251
Using Plot Ratios 253
Exporting Plots and Histograms 256
Configuring Instrument Settings 257
PMT and Laser Controls 258
Setting Up Multiple Panels 262
Running or Saving the Experiment 265
Running a Quick (Stat) Sample 266
Adding a Stat Tube to a Plate or Rack 267
Running an Existing Experiment 270
Importing Settings 272
Chapter 8 Acquiring Samples 275
Loading Sample Media 275
Setup Mode Controls 276
Acquiring Initial Sample in Setup Mode 278
Acquisition Mode Controls 282
Running Samples in Acquisition Mode 283
Using High-Throughput Mode 285
Pausing, Stopping, and Resuming Samples and Experiments 286
Pausing Sample Acquisition 286
Stopping Sample Acquisition 286
Using Pause After in an Experiment 286
Stopping and Resuming an Experiment 287
Exporting FCS and RLST Data Files 288
Exporting the FCSData File for a Single Position 288
Exporting FCS and RLST Data Files 290
Chapter 9 Applying Fluorescence Compensation 291
Setting Up the Experiment 291
Adjusting Compensation Automatically 295
Adjusting Compensation Manually 298
Dragging Populations 298
Editing the Compensation Matrix 299
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Table of Contents
Chapter 10 Analyzing, Saving, and Printing Data 301
File Types in Everest Software 301
File Structure of Saved Data 301
Analyzing Data 302
Loading Previous Experiments 303
Resuming Experiment Analysis 304
Analysis Toolbar 305
Working with Plots and Statistics in the Analysis Tab 306
Working with Compensation in the Analysis Tab 307
Exporting Data to CSV 308
Sending Analysis Settings to Acquisition 309
Publishing Data 309
Publish Toolbar 310
Preparing Reports 310
Printing a Report for the Selected Position 311
Printing a Report for All Positions 312
Chapter 11 Reports 313
Quality Control and ZE5-EYE Reports 313
Reports Tools 313
Generating Daily QC Reports 313
Generating QC Trending Reports 316
Generating ZE5-EYE Trending Reports 317
Generating User Reports 318
Chapter 12 Maintenance 319
Recommended Maintenance Schedule 319
Daily 319
Weekly 319
Monthly 320
Yearly 320
Cleaning Solutions 321
Disinfectants for Use in Sample Line 321
Disinfectants for Use in Sheath Line 321
Disinfectants for Use in the Waste Bottles 321
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Unclogging the Sample Line and Probe 322
Cleaning the Sample Line and Probe 324
Decontaminating the System 325
Required Materials 326
Preparing for the Decontamination Process 326
Decontaminating the System 328
Cleaning the Optical Filters 334
Replacing the QC Beads 335
Maintaining the System When Not in Use 338
Appendix A Troubleshooting 339
Exporting and Viewing Log Files 339
Deleting Acquired Data Files 340
Viewing Everest Software 2.0 FCSFiles in FlowJo Software 342
Fluidics Issues 343
Software Issues 343
Acquisition/Event Issues 344
Hardware/Electronics/Laser Issues 358
Appendix B Example 9-Color Immunophenotyping Experiment 359
Preparing Controls and Samples 359
Setting Up Fluorophores and Controls 360
Setting Up the Sample and Sampling Parameters 362
Creating Plots for the Experimental Sample 366
Acquiring Initial Data 368
Performing Initial Data Analysis 371
Performing Automatic Compensation 373
Resuming Acquisition 375
Analyzing or Exporting Final Data 376
Appendix C Optional External DI Water and Waste Ports 379
Connecting External DI Water 379
Connecting External Waste 381
Specifying External Port Use 381
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Table of Contents
Appendix D ZE5 Cell Analyzer Specifications 383
Appendix E Ordering Information 387
Appendix F References 389
Glossary 391
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Safety and Regulatory Compliance
For safe operation of the ZE5™ Cell Analyzer, Bio-Rad strongly recommends that you follow the safety
specifications listed in this section and throughout this guide.
Safety Warning Labels
Warning labels posted on the instrument and WARNING and Caution notes listed in this guide warn
you about sources of injury or harm. Review the meaning of each safety warning label.
Icon Explanation
Shock hazard! This symbol draws attention to a possible injury or danger to life if the
associated directions are not followed correctly. Only qualified, trained technicians should
carry out service work on electronic components, due to potential shock hazard.
Risque d'électrocution! Ce symbole met en garde contre les risque possibles de blessure
ou un danger pour la vie si les instructions fournies ne sont pas suivies correctement. Seuls
des techniciens qualifiés et formés peuvent effectuer des réparations sur des composants
électroniques en raison du risque d'électrocution.
Risk of danger! This symbol identifies components that pose a risk of personal injury or
damage to the instrument if the associated directions are not followed correctly. Wherever
this symbol appears, consult this guide for further information before proceeding.
Risque de danger! Ce symbole identifie les composants qui présentent un risque de
blessures ou de dommages pour l'instrument si les instructions ne sont pas suivies
correctement. Lorsque ce symbole apparaît, consultez ce guide pour plus d'informations
avant de continuer.
Table 1. Explanations of safety warning labels
User Guide | 13

Safety and Regulatory Compliance
Icon Explanation
Laser hazard! This symbol draws attention to a possible injury or danger to life due to laser
radiation if the associated directions are not followed correctly. Do not remove system
covers, which are in place for your safety. Only qualified, trained technicians should access
exposed laser beams.
Risque laser! Ce symbole attire l'attention sur un risque potentiel de blessure ou de danger
pour la vie dû au rayonnement laser lorsque les instructions fournies ne sont pas suivies
correctement. Ne retirez pas les caches de protection qui sont en place pour votre sécurité.
Seuls des techniciens qualifiés et formés doivent accéder aux rayons laser exposés.
Biohazard! Biosafety is of utmost importance while operating this instrument. This symbol
identifies components that may become contaminated with biohazardous material. When
handling biohazardous samples or the ZE5 Cell Analyzer’s waste container, adhere to the
recommended precautions and guidelines in this guide, and comply with any local guidelines
specific to your laboratory and location.
Risque biologique! La biosécurité est d'une importance capitale lors de l'utilisation de cet
instrument. Ce symbole identifie les composants qui peuvent être contaminés par des
matières à risque biologique. Lorsque vous manipulez des échantillons biologiques ou le
conteneur de déchets de l'analyseur de cellules ZE5, respectez les précautions et directives
fournies dans ce guide et respectez les directives locales spécifiques à votre laboratoire et à
votre site.
Consult documentation! This symbol identifies components for which operating
instructions must be followed to ensure safe and correct use. Wherever this symbol appears,
consult this guide for information before using the instrument component.
Consultez la documentation! Ce symbole identifie les composants pour lesquels les
instructions d'utilisation doivent être suivies pour assurer une utilisation sûre et correcte.
Chaque fois que cet instrument apparaît, consultez ce guide pour information avant d'utiliser
l'instrument.
Table 1. Explanations of safety warning labels, continued
14 | ZE5 Cell Analyzer and Everest Software

Safe Use Specifications and Compliance
Safe Use Specifications and Compliance
Laser Product Hazard Classification
The intent of the laser hazard classification is to identify hazards to users posed by the laser and to
provide appropriate protective measures. The ZE5 Cell Analyzer is a Class 1 laser product that
complies with 21 CFR 1040.10 and 1040.11, except for deviations pursuant to Laser Notice No. 50,
dated June 24, 2007 stating that operators are not exposed to harmful levels of laser radiation during
normal operation, maintenance, and/or service. During times of repair and/or major service by a
trained technician, laser safety controls for Class 3B lasers must be followed.
WARNING! Use of controls, adjustments, or procedures other than those specified
herein may result in hazardous laser radiation exposure.
Electrical Safety Information and Classification
The ZE5 Cell Analyzer conforms to international regulations encompassing the accessibility of high
voltages by the user (IEC61010-1). Use all protective housings and shields as specified in this guide.
Further information about specific electrical hazards is listed in the hardware description.
AC Fuse Requirements
Remove power cord before replacing fuses. Fuses are 5 x 20 mm and must be rated to AC250V, 4 A
slow blow such as Schurter 0034.3123.
AC Power Cord Requirements
Power cord must be IEC 60320-1 compliant with a C13 plug on the instrument end. If the power cord
must be replaced, replace it with only an adequately rated cord.
Position the instrument for easy access to the power switch and the power cord.
AC Outlet Requirements
Three outlets (instrument, monitor, computer), AC100–240V, 50–60 Hz, <500 W.
User Guide | 15

Safety and Regulatory Compliance
Regulatory Compliance
This instrument has been tested and found to be in compliance with all applicable requirements of the
following safety and electromagnetic standards:
nIEC 61010-1:2010 (3rd Ed), EN61010-1:2010 (3rd Ed). Electrical Equipment for Measurement,
Control, and Laboratory Use - Part 1: General Requirements
nUL/CSA 61010-1:2012 (3rd Ed), Standard for Safety Electrical Equipment for Electrical Safety
(USA, Canada, NRTL)
nIEC 61010-2-081:2015, EN61010-2-081:2015. Safety requirements for electrical equipment for
measurement, control and laboratory use. Part 2-081: Particular requirements for automatic and
semi-automatic laboratory equipment for analysis and other purposes (includes Amendment 1)
nIEC 60825-1:2014, EN 60825-1:2014. Safety of laser products - Part 1: Equipment classification
and requirements
nClass 1 laser product per IEC 60825-1 and CDRH requirements and regulations
nEN 61326-1:2013 (Class A) Electrical equipment for measurement, control and laboratory use.
EMC requirements, Part 1: General requirements
nIEC 61326-1:2012 (Class A) Electrical equipment for measurement, control and laboratory use.
EMC requirements - Part 1: General requirements
nFCC Part 15 Subpart B Emissions (Class A)
nThis ISM device complies with Canadian ICES-001
nCet appareil ISM est conforme a la norme NMB-001
This equipment generates, uses, and can radiate radio frequency energy and, if not installed and
used in accordance with the instruction manual, may cause harmful interference to radio
communications. Operation of this equipment in a residential area is likely to cause harmful
interference, in which case the user will be required to correct the interference at their own expense.
Hazards
The ZE5 Cell Analyzer is designed to operate safely when used in the manner prescribed by the
manufacturer. If the ZE5 Cell Analyzer or any associated component is used in a manner other than
prescribed, or if modifications to the instrument are not performed by a Bio-Rad or other authorized
agent, then the warranty on the system will be voided and the protection provided by the equipment
might be impaired. Service of the ZE5 Cell Analyzer should be performed only by Bio-Rad personnel.
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Hazards
Biohazards
The ZE5 Cell Analyzer is a laboratory product. However, if biohazardous samples are present, adhere
to the following guidelines and comply with any local guidelines specific to your laboratory and
location.
General Precautions
nAlways wear laboratory gloves, coats, and safety glasses with side shields or goggles.
nKeep your hands away from your mouth, nose, and eyes.
nCompletely protect any cut or abrasion before working with potentially infectious materials.
nWash your hands thoroughly with soap and water after working with any potentially infectious
material before leaving the laboratory.
nRemove wristwatches and jewelry before working at the bench.
nStore all infectious or potentially infectious material in unbreakable leak-proof containers.
nBefore leaving the laboratory, remove protective clothing.
nDo not use a gloved hand to write, answer the telephone, turn on a light switch, or touch anything
that other people may touch without gloves.
nChange gloves frequently. Remove gloves immediately when they are visibly contaminated.
nDo not expose materials that cannot be properly decontaminated to potentially infectious material.
nUpon completion of the operation involving biohazardous material, decontaminate the work area
with an appropriate disinfectant (for example, a 1:10 dilution of household bleach).
Disposal of Biohazardous Material
The ZE5 Cell Analyzer includes a waste container that may potentially contain hazardous biological
materials, depending on the sample used. Dispose of the following potentially contaminated materials
in accordance with laboratory, local, regional, and national regulations:
nContent in waste container
nReagents
nUsed reaction vessels or other consumables that may be contaminated
Chemical Hazards
The ZE5 Cell Analyzer includes a waste container that may potentially contain hazardous chemical
materials, depending on the sample used.
User Guide | 17

Safety and Regulatory Compliance
Explosive or Flammability Hazards
The ZE5 Cell Analyzer system poses no uncommon hazard related to flammability or explosion when
used in a proper manner as specified by Bio-Rad Laboratories.
Electrical Hazards
The ZE5 Cell Analyzer poses no uncommon electrical hazard to operators when installed and
operated properly without physical modification and if connected to a power source of proper
specification.
Transport
Moving the ZE5 Cell Analyzer is not recommended after installation. If the system must be moved,
follow the decontamination procedure in this guide and remove all bulk fluidics. After a move, you
must run the QC process to ensure that the instrument is functioning properly.
Caution: Lift the instrument with the inset handles on the base. To reduce the risk of
personal injury or damage to the instrument, a minimum of two people must perform
this task. Use caution to keep the instrument level, and handle the instrument gently.
Operating Conditions
The ZE5 Cell Analyzer must be operated under the following conditions:
nTemperature range 18–25°C
nRelative humidity 20–60%
Disposal
The ZE5 Cell Analyzer contains electronic or electrical materials; they should be disposed of as
unsorted waste and must be collected separately, according to European Union Directive 2002/96/CE
on waste and electronic equipment — WEEE Directive. Before disposal, contact your local Bio-Rad
representative for country-specific instructions.
Warranty
The ZE5 Cell Analyzer and associated accessories are covered by a standard Bio-Rad warranty.
Contact your local Bio-Rad Laboratories office for details of the warranty.
18 | ZE5 Cell Analyzer and Everest Software