BIO RAD ZE5 User Manual Download Page 293

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Chapter 9

Applying Fluorescence Compensation

Everest™ Software offers simplified workflows for both manual and automatic compensation. To

perform automatic compensation, acquire single-color controls using the Everest compensation

template. You can then adjust regions to define the positive populations in all controls’ channels and

initiate the automatic compensation calculation. Everest Software also offers automatic region

determination, so that you do not need to manually adjust the gates to define the positive populations.

Tip:

Everest Software permits visualization of both compensated and uncompensated data in the

same workspace. Be sure to select the Comp parameter for plot axes so that the compensation

matrix is applied to the data.

For information on applying compensation to multicolor experiments, see

Appendix B, Example 9-

Color Immunophenotyping Experiment

Setting Up the Experiment

Before applying compensation in Everest Software (either automatically or manually), set up the

workspace to streamline the compensation process. For information about the Compensation

template, see

Setting Up Compensation Controls on page 182

Set up an experiment by selecting fluorophores and activating detectors as described in

Chapter

7, Creating Experiments and Workspaces

User Guide | 291

Summary of Contents for ZE5

Page 1: ...ZE5 Cell Analyzer and Everest Software User Guide Version 2 0 ...

Page 2: ......

Page 3: ...ZE5 Cell Analyzer and Everest Software User Guide Software Version 2 0 ...

Page 4: ...r 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 ...

Page 5: ...y 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 Sy...

Page 6: ...ilters 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 You...

Page 7: ...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 Preferenc...

Page 8: ...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 Pl...

Page 9: ...n 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 Plo...

Page 10: ...uiring 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 Dat...

Page 11: ...ng 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 Mai...

Page 12: ...uidics 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 37...

Page 13: ...Table of Contents Appendix D ZE5 Cell Analyzer Specifications 383 Appendix E Ordering Information 387 Appendix F References 389 Glossary 391 User Guide xi ...

Page 14: ...Table of Contents xii ZE5 Cell Analyzer and Everest Software ...

Page 15: ...cution 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...

Page 16: ...d 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 analyse...

Page 17: ...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 hardw...

Page 18: ... Part 1 General requirements n IEC 61326 1 2012 Class A Electrical equipment for measurement control and laboratory use EMC requirements Part 1 General requirements n FCC Part 15 Subpart B Emissions Class A n This ISM device complies with Canadian ICES 001 n Cet appareil ISM est conforme a la norme NMB 001 This equipment generates uses and can radiate radio frequency energy and if not installed an...

Page 19: ...o write answer the telephone turn on a light switch or touch anything that other people may touch without gloves n Change gloves frequently Remove gloves immediately when they are visibly contaminated n Do not expose materials that cannot be properly decontaminated to potentially infectious material n Upon completion of the operation involving biohazardous material decontaminate the work area with...

Page 20: ...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 n Temperature range 18 25 C n Relative humidity 20 60 Disposal The ZE5 Cell ...

Page 21: ... fluorescence compensation a fluorophore selection panel and a run list design wizard The ability to analyze files while acquiring new data saves time and streamlines your workflow Key features include n Integrated programmable wash station to reduce sampling time and sample carryover n Onboard calibration beads for rapid QC without user intervention n Volumetric sample uptake for absolute countin...

Page 22: ...tallation Requirements The ZE5 Cell Analyzer should be installed by a Bio Rad service engineer to ensure proper instrument operation and calibration If any items are missing or damaged contact your local Bio Rad office Choose an appropriate site for ZE5 Cell Analyzer installation such as a sturdy bench or tabletop away from any other instruments that might interfere electrically or mechanically by...

Page 23: ...r n Updating the ZE5 instrument firmware Tip You can upgrade Everest Software versions 1 2 2 1 to version 2 2 Upgrading Everest Software Important The software upgrade process removes the currently installed version of Everest Software Before starting the installation process ensure that no experiments are running on the instrument and that you have saved all data and exited the software Note You ...

Page 24: ... window and then the ZE5 Service Tool dialog box appear on the screen Do not close the dialog box or shut down the instrument during the update To update the ZE5 instrument firmware 1 Start Everest Software on the Everest computer If the firmware update is required Everest Software displays the ZE5 Firmware Update dialog box 2 Click Update firmware The Everest Firmware Update Utility window appear...

Page 25: ...C reports ü ü Generate QC trending and EYE trending reports ü Acquire samples ü ü Generate analysis reports ü ü Clean probe and sample line ü ü Decontaminate system ü ü Edit QC criteria ü Configure global instrument and software preferences ü Create users ü Deactivate users ü Edit users ü Change user rights ü Reset other users passwords ü Generate user reports ü Change own password ü ü Table 3 Fea...

Page 26: ...is not required To access training modules 1 Click the Main Menu button in the upper right corner formerly known as the Help and Information menu button this button is depicted by three green bars 2 Select Training Module 3 In the Training Modules dialog box click the link to the training module you want to view The training module opens in the browser To access this user guide from Everest Softwa...

Page 27: ...scription Read this section to understand the ZE5 Cell Analyzer system hardware before operating the instrument System Overview The ZE5 Cell Analyzer system consists of fluidics optics electronics and software User Guide 25 ...

Page 28: ...ntrol of the instrument Caution Be careful not to trip on the power cord or Ethernet cable when you walk around the instrument n Power switch press the main power switch to turn on power to the system Caution Do not use the main power switch to shut down the system Shut down the system using Everest Software For more information see Shutting Down on page 161 This area of the instrument also contai...

Page 29: ...ed information about the hydrodynamic focusing that occurs in the flow cell see Flow Cell on page 33 Four large 4 L and two small 450 ml bottles are located in the instrument s bulk fluidics chamber The two large bottles with blue caps contain DI water the two large bottles with red caps collect waste Mounted below the four large bottles the small bottle with the blue cap contains system cleaner a...

Page 30: ...een runs These bottles have a blue cap Each holds 4 L of fluid together they provide about 8 hr of continuous run time Note When refilling these bottles ensure that the tubing and filter are reinstalled so that they are in the lower corner of the bottle opposite the cap Sheath Additive Bottle The sheath additive bottle which has a white cap contains 250 ml of concentrated balanced salts antimicrob...

Page 31: ...ves the integrity of the system For more information see n Shutting Down on page 161 n Cleaning Solutions on page 321 Fluidics Filters All of the onboard reagents are filtered through 0 2 µm capsule filters that remove particulates from the fluid before it is circulated through the system This helps reduce background noise especially in the scatter channels and helps prevent microbes from entering...

Page 32: ... ensure proper handling and disposal of biohazardous substance You can add 400 ml of full strength bleach to an empty waste bottle upon installation in the instrument for a final concentration of 10 in the full waste bottle Alternatively you can add full strength bleach to a full waste bottle and allow it to sit to thoroughly decontaminate biohazardous material in the waste Fluidics Connections Th...

Page 33: ... Examples are shown in the next figure For information about connecting the DI water and waste bottles to house DI water and waste respectively see Appendix C Optional External DI Water and Waste Ports Sample Loader The ZE5 Cell Analyzer features a sophisticated autoloader that can sample from a wide variety of media types including racks of forty 5 ml tubes 12 x 75 mm twenty four 1 5 ml tubes 96 ...

Page 34: ...c sample pump that delivers sample to the interrogation points in the flow cell Using Everest Software you can adjust sample target flow rates within the range of 0 1 3 5 µl sec 6 210 µl min The pump can also run in reverse to deposit sample back into a tube to deposit reagent from another position into a sample position or to clear blockages from the sample line An automated door slides up to pro...

Page 35: ...he flow cell is the heart of the system and is where sample interrogation occurs It is composed of fused silica that surrounds a 145 x 265 µm channel through which sheath fluid flows and focuses the sample fluid Sample is drawn up into the sample line by the sample pump and is introduced into the flow cell through a sample introduction needle Sheath fluid is introduced under pressure in an upward ...

Page 36: ...ent to a waste container Fluidics connections to the flow cell area are shown in the next figure The fluidics system allows sheath fluid flow within the flow cell to be reversed to help remove blockages Cleaning fluid is introduced into the flow cell as part of the system shutdown process 34 ZE5 Cell Analyzer and Everest Software ...

Page 37: ...n the flow cell they also focus and filter the laser light before it reaches the detectors The PMTs detect scattered and fluorescent light signal Lasers The ZE5 Cell Analyzer can be configured with a selection of up to five lasers from the Coherent OBIS line of lasers A typical configuration contains lasers of wavelengths and powers as follows n 355 nm UV at 50 mW n 405 nm violet at 100 mW User Gu...

Page 38: ...correct dimensions and focus Consistent geometry between lasers optimizes illumination of each analyzed cell and maintains a high degree of measurement precision Interrogation An interrogation point occurs where each laser beam intercepts the sample in the flow cell The ZE5 Cell Analyzer system supports up to five spatially separated interrogation points along the core stream Upon interrogation th...

Page 39: ...ultiple particles passing through an interrogation point The ZE5 Cell Analyzer can include up to two FSC detectors Both are highly sensitive PMTs with adjustable voltage The default FSC detector measures 488 nm light from 2 18 relative to the laser beam It can resolve cells from debris and measure particles from 0 5 50 μm in diameter Typical uses include generation of plots of lysed whole blood su...

Page 40: ...imize the collected light on all detectors guaranteeing that the light efficiently reaches the final bandpass filter with no more than three interactions with dichroic filters Mirrors and filters permit multiparametric analysis By partitioning the spectrum of collected light into specific ranges of wavelengths each detector can be dedicated to the measurement of particular fluorophores The ZE5 Cel...

Page 41: ...n be dichroic or normal incidence Dichroic filters reflect light that is not permitted to pass through them They are typically placed at a 45 degree angle to the incident light and are used to direct light around the detection path Normal incidence longpass filters and bandpass filters typically absorb light that is not permitted to pass through them They are placed directly in front of detectors ...

Page 42: ...ensity of all wavelengths of light equally by reflecting or absorbing a portion of it For experimental applications where events appear off scale when the detector is set at minimum gain a neutral density filter can attenuate the signal and keep the events on scale In the ZE5 Cell Analyzer neutral density filters are used in front of the FSC detector You also have the option to install neutral den...

Page 43: ...5 Cell Analyzer can be configured with up to five banks one for each laser The five banks are grouped into three regions A B and C Region A located on the bottom consists as a single bank while regions B and C located on the top left and top right respectively each contain two banks of PMTs Bank Laser Total possible PMTs A Blue 488 nm 7 B Top Red 640 nm 5 B Bottom UV 355 nm 5 Table 5 Optical filte...

Page 44: ...Chapter 2 Hardware Description Bank Laser Total possible PMTs C Top Violet 405 nm 7 C Bottom Yellow Green 561 nm 7 Table 5 Optical filter banks continued 42 ZE5 Cell Analyzer and Everest Software ...

Page 45: ...ors and filters are configured in a combination of fixed and operator changeable components Each bank contains an initial array of fixed dichroic mirrors followed by replaceable dichroic mirrors and filters User Guide 43 ...

Page 46: ... are installed in region A while double filter holders are installed in stacked banks regions B and C Examples of single and double filters are shown in the next figure For information about configuring filters see Working with Optical Filter Configurations on page 120 44 ZE5 Cell Analyzer and Everest Software ...

Page 47: ...5 EYE uses multiple LEDs to pulse ten different wavelengths of light into the optical filters that lead to the detector banks These LEDs are shown in the next figure The ZE5 EYE process is a component of the QC process and is run any time the QC process is initiated For more information see Using the ZE5 EYE to Confirm Filter Choices on page 135 User Guide 45 ...

Page 48: ...n in the software can be updated accordingly For more information about the ZE5 EYE see Using the ZE5 EYE to Confirm Filter Choices on page 135 Photomultiplier Tubes PMTs detect and amplify the scattered and fluorescent light signals produced by laser interrogation of the particles Located behind the optical filters the PMTs detect specific bands of fluorescent light based on the attached fluoroch...

Page 49: ...Everest Software for analysis WARNING Shock hazard Due to potential shock hazard only qualified trained technicians should carry out service work on electronic components Pre Amplifiers Pre amplifiers boost the signals coming from the PMTs Analog to Digital Converters Analog to digital converters ADCs convert the electrical signal coming from the pre amplifier into a digital signal and transfer th...

Page 50: ...Chapter 2 Hardware Description 48 ZE5 Cell Analyzer and Everest Software ...

Page 51: ... To open the loader door and extend the loader press the silver sample chamber button on the front of the instrument The external light turns on when the loader door is opened The sample chamber button also controls the inner loader chamber light when the door is closed Press and hold the button to turn the internal illumination on or off A wash module is integrated into the sample delivery system...

Page 52: ...ntinuously between samples drawing air and wash fluid into the sample line between samples these air bubbles and wash fluid segments serve as sample separators Loader Components The various components of the loader are depicted in the next figure and described in the accompanying table 50 ZE5 Cell Analyzer and Everest Software ...

Page 53: ...livery to the flow cell 6 Rinse line and rinse waste line Introduces sheath fluid to the wash station for cleaning the probe and sample line During certain stages of instrument operation such as shutdown cleaning fluid rather than sheath fluid can be used for washing 7 Gauge Supports the sample probe and the wash station 8 Wash station Facilitates probe and sample line cleaning 9 Bead station Hous...

Page 54: ...tes The ZE5 Cell Analyzer includes a bi level tube lifter shown in the next figure When a full 40 tube rack is placed on the tool tubes in every other row are slightly raised as shown in the next figure This facilitates tube removal and replacement during sample preparation 52 ZE5 Cell Analyzer and Everest Software ...

Page 55: ...ted portion of the sample probe up and down through the wash station The inside of the sample line is cleaned by moving the probe up and down through the wash station while the sample pump is running This method introduces air and sheath segments into the sample line and pushes residual sample and debris off the walls and through to waste This results in highly effective cleaning and minimal carry...

Page 56: ...5 Loader Combined with photo sensors in the loader the gauge helps ensure that the vertical travel distance of the probe and wash station is appropriate for the media type 54 ZE5 Cell Analyzer and Everest Software ...

Page 57: ...gin window n Home window n Help and Information menu n Recent Experiment Sessions panel n Experiment Builder guides you through experiment setup using four sequential screens o Name o Fluorophores o Samples o Workspace Settings n Acquisition workspace n Instrument Control panel n Toolbar n Status bar For information about which sections of the Everest Software user interface are available only to ...

Page 58: ...logged in user s name Details displays fluidics and system status as well as software and firmware version information Shutdown displayed if the system has been started up The instrument can be shut down without user login as long as samples are not actively running See Shutting Down on page 161 Startup displayed if the system has not yet been started up The instrument can be started up without us...

Page 59: ...stem administrator Password Enter the password for this user name Notes Enter any notes for this session Notes appear in the user reports available to administrators Table 7 User login items and their functions Home Window The Home window appears after you log in to the system User Guide 57 ...

Page 60: ... allows you to initiate experiments in various ways Button Function Launches the Experiment Builder which guides you through setup of fluorophores detectors samples workspace and plots Skips the Experiment Builder and allows you to run a sample quickly from the single tube position in the loader By default all lasers and all parameters are active when acquiring sample in stat tube mode Loads a pre...

Page 61: ...per right corner of the Everest Software window You can access these items at any time including when no user is logged in Button Function Log Extraction pulls system log files from the last 180 days compresses them into a ZIP file and saves the file on the computer s desktop Training Module displays a list of training videos that guide you through basic ZE5 Cell Analyzer system operations These v...

Page 62: ...allows you to reset your login password Available only to nonadministrators Manage Users allows you to manage login accounts and access rights Available only to administrators User Report opens a report that tracks usage over time and includes session notes entered by logged in users Available only to administrators Global Preferences allows you to configure global settings for the instrument and ...

Page 63: ...ministrative rights the Reset Password button appears in the Home window tools above the Recent Experiment Sessions panel To reset your password 1 Click Reset Password 2 In the Reset Password dialog box enter the new password twice and click OK User Guide 61 ...

Page 64: ... a single experiment by clicking the up arrow next to its name Load Run List allows you to browse for and load experiment files that are stored in locations other than the default recent experiments folder D EverestUsers username You must know the name of the rlst file that you seek Resume Loads the run list as it was last acquired into the workspace Previously acquired positions are indicated in ...

Page 65: ...ons The Home window includes the following quick action items that appear depending on the state of the instrument Quick action area Item Function QC initiates the QC process See Running Quality Control on page 156 Shutdown displayed if the system has been started up See Shutting Down on page 161 Startup displayed if the system has not yet been started up See Starting Up the System on page 152 Tem...

Page 66: ... choosing the appropriate detection channels Samples Facilitates configuration of the sample media required for the experiment as well as the acquisition settings for each control and sample Workspace Settings Facilitates setup of the workspace where you can create plots enable lasers adjust PMT voltages and set the trigger threshold as needed Table 13 Experiment Builder screens Name Screen The Na...

Page 67: ...Note If the ZE5 Cell Analyzer is configured with a large number of PMTs enabling all detectors can create large data files that can be difficult to handle upon export to other software packages for analysis Cancels setup of this experiment Returns you to the Home window where you can start a new experiment Returns to the previous Name screen Moves to the next Samples screen Table 14 Fluorophores s...

Page 68: ... Use the search box to quickly find a fluorophore by typing in the first few letters of its name Item Function Hides fluorophores that are not used regularly These settings apply to the logged in user s profile the selected fluorophores will not be hidden from other users Restores the fluorophore list to the default list delivered with Everest Software Table 15 Select Fluorophore s panel items and...

Page 69: ... with it Because multiple dyes are excited with the same laser spectra are overlaid on the same graph When you select a fluorophore its emission spectrum is shown in the graph associated with the laser that optimally excites it Additionally the gray regions in a graph represent the wavelengths allowed through by the bandpass filters located in the laser s fluorescence detection paths User Guide 67...

Page 70: ... needed Tip The only parameters that are activated are those for which fluorophores have been selected Therefore only data from the enabled active parameters will be collected during acquisition Height 24 bit resolution area 24 bit resolution and width 17 bit resolution using linear interpolation at half height are collected for all active parameters All signals are collected as raw digital values...

Page 71: ...m Function Cancels setup of this experiment Returns you to the Home window where you can start a new experiment Returns to the previous Fluorophores screen Moves to the next Settings screen Table 16 Samples screen items and their functions Media Selector Before you can set up the sample positions you must select the type of media to contain the samples User Guide 69 ...

Page 72: ...l plate 96 deep well plate 384 well plate Configure a custom device from which sample can be acquired can also be used to calibrate a standard plate Table 17 Media selector buttons and their functions After you select the media type the full Samples screen appears The Samples screen contains three panels n Selected Fluorophores n Plate Setup n Sample Naming 70 ZE5 Cell Analyzer and Everest Softwar...

Page 73: ...rophores that were selected on the previous screen The Template area of the Selected Fluorophores panel allows you to select a pulse parameter for compensation add single color compensation controls to the first wells or tubes designate a negative control and designate a universal negative control for the experiment User Guide 71 ...

Page 74: ...Settings Controls on page 179 For information on controls in the Plate Setup area that are not covered in this chapter see Position Settings Controls on page 193 Selected Media Type The selected media type pane allows you to program each well or group of wells with a set of run conditions The next figure shows the currently selected well highlighted with a red border 72 ZE5 Cell Analyzer and Evere...

Page 75: ...Experiment Builder If multiple wells are selected the red border surrounds each of them as shown in the next figure User Guide 73 ...

Page 76: ...e of the following n To select a well click it n To select multiple adjacent wells click the first well and drag over the target wells n To select multiple nonadjacent wells press and hold Ctrl and click the wells 74 ZE5 Cell Analyzer and Everest Software ...

Page 77: ...tting is OFF Allows you to assign a name to a well The FCS file that is saved for the well includes this name Displays the assigned name of the selected Plate Map position Table 18 Selected Media items and their functions Position Types Below the plate map the Type setting allows you to designate a sample position or group of sample positions as Sample Setup Wash or Reagent Button Function Appears...

Page 78: ...applying automatic compensation Table 20 Images used on sample positions High Throughput Mode There are two ways to run samples standard mode and high throughput mode In the default standard single sample Acquisition mode each sample is acquired as an independent run The sample is boosted to the flow cell before acquisition begins and there is only one sample in the sample line at any given time S...

Page 79: ...are available only in standard sampling mode High throughput mode Standard mode Number of samples tubes or wells in sample line during run Multiple One Boost performed before sample acquisition No Yes Return Sample function allowed No Yes Pause between samples function allowed No Yes Wash between samples allowed Yes Yes Agitation allowed Yes Yes Event limit allowed No Yes Gate limit allowed No Yes...

Page 80: ...sing the arrow button in the upper right corner In addition to Location Name and Sample Type the expanded run list shows the following information for each position target Flow Rate target Event Rate Max Volume for the media type sample Volume Limit Event Limit Agitation Time added Reagent Position Reagent Volume Probe Wash Times outside and inside Acquisition mode High Throughput versus Standard ...

Page 81: ...fy the details of each sample by scrolling across the run list The Multiple tab facilitates naming multiple samples at a time using components including Date Well ID Sequence ID Experiment Name and Custom text For more information see Labeling Positions Automatically on page 184 User Guide 79 ...

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Page 83: ...Experiment Builder The Keyword tab allows you to create custom keyword value pairs and associate them with sample positions For more information see Setting Up Keywords on page 190 User Guide 81 ...

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Page 85: ...and Histogram Tools on page 217 n Set initial PMT voltages if known from a previous experiment or specifically for this particular sample type For information about setting PMT voltages see PMT Control Panel on page 85 and Configuring Instrument Settings on page 257 n Configure initial trigger and threshold values if known from a previous experiment or specifically for this particular sample type ...

Page 86: ...his experiment and returns you to the Home window where you can start a new experiment Table 22 Settings screen items and their functions Plate Map The plate map in the Settings screen displays the layout of the plate before the run list has been applied to the Acquisition workspace The plate map in the Settings workspace displays different colors and symbols for each well position Symbol Descript...

Page 87: ...position Unassigned position Table 23 Plate map symbols continued PMT Control Panel The PMT Control panel in the Settings workspace allows you to control initial settings related to lasers and detectors These settings can be adjusted later after acquisition begins User Guide 85 ...

Page 88: ...e Legend 1 Toggle between detector filter numbers and detector names 2 Load PMT voltages from a selected run list file 3 Reset PMT voltages to default values 4 Enable and disable lasers 86 ZE5 Cell Analyzer and Everest Software ...

Page 89: ...ltiple experiments by adding panels in the Experiment Builder Adding a panel returns you to the Experiment Builder Name screen After assigning a unique name you can set up a unique run list in a portion of the plate map The name of each panel in the experiment is shown at the bottom of the Settings screen as shown in the next figure Groups of positions in the plate map can have uniquely defined se...

Page 90: ...STAT Tube option in the Home window you bypass the Experiment Builder Instead the Acquisition workspace opens in which you can create plots add gates and adjust settings You can acquire samples and save data directly from this screen In stat tube mode all parameters are activated by default and can be named by directly modifying the name in the parameter list 88 ZE5 Cell Analyzer and Everest Softw...

Page 91: ...Saving and Printing Data n Publish provides tools for printing reports Allows you to arrange plots so that you can export them to PDF or other applications such as PowerPoint For more information see Chapter 10 Analyzing Saving and Printing Data Instrument Control Panel The Instrument Control panel located on the left side of the Acquisition workspace displays sampling controls for setup and Acqui...

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Page 93: ...ou use Setup mode in this way sampling does not stop until you manually stop the run 2 You can use the Record function to record data files from samples sampling stops when the preset limit is reached This is useful if you need to acquire and save data from samples on a single tube basis These data are saved to an FCS file which you can later open and review You can use the Record function for som...

Page 94: ...e acquisition has been initiated in Acquisition mode the ZE5 Cell Analyzer proceeds to each position on the rack or plate starts and stops sampling as specified in the run list and records data files Acquisition mode requires minimal user intervention after acquisition has begun For more information see Acquisition Mode Controls on page 282 and Running Samples in Acquisition Mode on page 283 92 ZE...

Page 95: ...bar items and their functions Tools The following tools appear in both the Settings screen toolbar and the Acquisition tab toolbar unless otherwise noted Button Function Home returns you to the Home window where you can create a new experiment or open one that has already been created Note This tool appears only in the Acquisition workspace toolbar Advanced Plot Builder facilitates creation of his...

Page 96: ...etection banks Also allows you to initiate the ZE5 EYE process Note This tool appears only in the Acquisition workspace toolbar Export allows you to select from five export options n Export FCS file for a single position n Export all FCS files for the current experiment n Export most recent FCS file for each position and compress to ZIP n Export run list to RLST format and export all FCS files for...

Page 97: ...lows you to specify a gate limit Apply the gate limit to experimental samples as opposed to compensation controls Apply the gate limit to the selected sample positions Table 26 Batch toolbar tools and their functions Quick Action and System Action Tools A quick action tool is available in the Acquisition tab toolbar Button Function Analyze opens the most recent run list in the analysis tab This is...

Page 98: ...mber Light turns the loader chamber light on and off Home Loader returns the loader to its home position See Accessing the Loader on page 159 Clean runs cleaner through the probe and sample line See Cleaning the Sample Line and Probe on page 324 Unclog moves the probe to the port behind the stat tube position in the sample loader and cycles through the unclog protocol See Unclogging the Sample Lin...

Page 99: ...be replaced soon Extremely Low Fluidics Warning indicates that the bulk fluidics should be immediately replaced Swap Fluidics switches the active waste and sheath fluidics bottles See Refilling Bulk Fluidics on page 143 Bottle Use Indicator indicates whether the top or bottom waste and DI water bottles are in use Waste Fluid Level indicates the level of fluid in the waste bottles DI Water Level in...

Page 100: ...ure is set in the Experiment Builder for a particular experiment that value overrides any previous number set in the Home window Table 29 System buttons and their functions continued Other Toolbar Buttons Buttons in the toolbar vary depending on the stage of experiment setup and the selected tab n For information about buttons for reports see Reports Tools on page 313 n For information about toolb...

Page 101: ...dds a window that displys a log of all error warning and information notifications that have been issued since the software was started Note This log window appears only in the Acquisition workspace 6 Displays the most recently issued instrument status warning Status Description Starting Up The system is performing the startup process Calibrating The system is running the QC process Ready The syst...

Page 102: ...pplies only to creating moving resizing or deleting a region creating moving resizing or deleting a plot and applying or removing a gate 8 Redo reverses the last Undo action 9 Clear removes plots and histograms that have been added to the workspace Notifications and System Logs Everest Software displays error warning and information notifications in the lower right area of the screen after you log...

Page 103: ...software as well as actions taken by users and instrument functions initiated by Everest Software System logs can provide useful troubleshooting information needed by Bio Rad Technical Support You can use the Help and Information menu to extract detailed system log information to a ZIP file For information about log files see Exporting and Viewing Log Files on page 339 User Guide 101 ...

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Page 105: ...ysical filter changes n Reverting the optical filter configuration to factory settings Tip Both administrators and nonadministrators can change the physical optical filters Managing Users As an administrator you can use the Manage Users dialog box to create new users 0deactivate existing users manage user passwords set user file storage directories and set user privileges The User Management butto...

Page 106: ...m To add a user account 1 In the Home window click Manage Users The User Management dialog box opens 2 Click Add User The Add user dialog box opens By default the Active checkbox is selected 104 ZE5 Cell Analyzer and Everest Software ...

Page 107: ...Enter a user name 5 Do one of the following n To require that users set their own password a In the Password text box type a default password For example Change Password b Type it again a Select Require password reset b Give the default password to the user User Guide 105 ...

Page 108: ...c Give the password to the user 6 Select Standard or Administrator to set the account type 7 In the Directory area click Select to specify a folder in which the user s instrument data will be saved Note Any path specified here takes precedence over the default file save path set in the global preferences For more information see Setting File Save Parameters on page 115 8 Type the first and last na...

Page 109: ...Managing Users 9 Click OK to save the new user account User Guide 107 ...

Page 110: ... been created you can edit details such as password email address default file directory and user rights To edit user information 1 In the User Management dialog box click the pencil button for the user The Edit user dialog box opens 108 ZE5 Cell Analyzer and Everest Software ...

Page 111: ... area takes precedence over the default file save path set in the global preferences For more information see Setting File Save Parameters on page 115 3 Click OK Tip If all administrator passwords are lost or forgotten call Bio Rad Technical Support to receive a temporary administrator password User Guide 109 ...

Page 112: ... user respectively 2 To change system access rights for a user select Standard or Admin 3 Select Show inactive users to view inactive user accounts clear it to hide inactive user accounts 4 Click Save Setting Global Preferences Administrators can set global preferences for the ZE5 Cell Analyzer and Everest Software The Global Preferences button is available in the Home window to users with an Admi...

Page 113: ...ample runs out n Set default QC criteria n Set permissions for startup shutdown and QC when a user is logged out n Set default file save parameters n Specify number of experiment sessions to load n Display or hide button text in the software interface n Enable vacation mode n Specify how plot statistics are displayed n Reset the system to default settings User Guide 111 ...

Page 114: ...l Port Use on page 381 System sleep time settings are reserved for future use Setting Plot Display Defaults and Stopping Acquisition In the Acquisition and Plots area of the global preferences you can determine the size of plots added to the workspace specify whether off scale data indicators appear in plots and set the system to stop acquisition when sample runs dry To set plot display defaults 1...

Page 115: ... figure might indicate the need to adjust instrument settings 3 Select a resolution setting for new plots 4 Select the Sample dry checkbox if you want to stop acquisition when bubbles occur indicating that sample has run out 5 Click OK to save the changes User Guide 113 ...

Page 116: ... required to place the population in the center of the histogram must be less than this value n Max CV Increase the maximum increase in the calculated coefficient of variation CV measured from baseline for each parameter must be less than this value To edit the QC criteria 1 In the Home window click Global Preferences 2 In the QC Criteria area adjust the values for QC Max Voltage Change and QC Max...

Page 117: ...es when they try to save files that exceed a certain threshold To set file save parameters 1 In the Home window click Global Preferences 2 In the File Save Parameters area click Select navigate to the target location for saving FCS files and click OK The location appears in the File Save Path 3 Use the slider to specify the threshold for the Minimum disk space warning If the specified minimum disk...

Page 118: ...wn arrow next to the experiment name in the Home window 3 Select the Show Button Text checkbox to display button text clear it to hide button text 4 Click OK to save the changes Setting Up Vacation Mode To maintain optimal system performance Bio Rad recommends that you run the ZE5 Cell Analyzer regularly without long periods of disuse If your instrument will not be in use for an extended period yo...

Page 119: ...at will occur during the vacation period To set up vacation mode 1 In the Home window click Global Preferences 2 Select the Enable Vacation Mode checkbox 3 Set the vacation date range by selecting dates from the start date and end date calendars After the end of the vacation period vacation mode automatically resets to off 4 Specify how often and at what time of day the system startup should occur...

Page 120: ...Chapter 5 Configuring the System 118 ZE5 Cell Analyzer and Everest Software ...

Page 121: ...l Preferences 2 In the Statistics area select the checkbox for each statistic that you want to show and clear the checkbox for each statistic that you want to hide 3 Assign a unique number to each statistic to specify the display order The selected statistics appear in ascending order those with higher numbers appear to the right of those with lower numbers 4 Click OK to save the changes User Guid...

Page 122: ...ent physical filter you must edit the corresponding mirror filter position labels in this section of Everest Software see Editing the Optical Filter Configuration on page 132 A green check mark on each final position in each optical detection path indicates that the ZE5 EYE process has verified that the filter listed in Everest Software is currently installed in the instrument The ZE5 EYE relies o...

Page 123: ...of the 488 nm laser Labels A1 through A9 show the filters that you can change Any changes that you make to the labels here will propagate to the parameter labels in other parts of Everest Software Note After you save changes to the optical filter configuration in the software the ZE5 EYE process runs to confirm that the software changes match the physical filter setup Depending on the laser setup ...

Page 124: ...Chapter 5 Configuring the System Detection paths for banks B and C are shown in the next four figures 122 ZE5 Cell Analyzer and Everest Software ...

Page 125: ...Working with Optical Filter Configurations User Guide 123 ...

Page 126: ...Chapter 5 Configuring the System 124 ZE5 Cell Analyzer and Everest Software ...

Page 127: ...Working with Optical Filter Configurations The following detection paths can be viewed from the Optical Filter Configuration dialog box but not edited n FSC1 n FSC2 User Guide 125 ...

Page 128: ...ollowing sections show some standard combinations of optical elements that are available 355 nm UV Laser Filter Choices UV 355 nm 50 mW 5 Detectors Fluorophores 387 11 BUV395 Indo 1 hi Ca2 saturated 405 DLP changeable 447 60 Alexa Fluor 350 DAPI PureBlue Hoechst 33342 Indo 1 lo Ca2 free 493 DLP 525 50 BUV496 BUV563 560 DLP 670 30 BUV661 Hoechst 33342 690 DLP changeable 700 LP BUV737 126 ZE5 Cell A...

Page 129: ...0 mW 7 Detectors Fluorophores 420 10 Cascade Blue BV421 439 DLP changeable 460 22 Pacific Blue BD Horizon V450 493 DLP 525 50 AmCyan BV510 Cascade Yellow Pacific Orange 581 DLP changeable 615 24 BV605 640 DLP 670 30 BV650 690 DLP 720 60 BV711 752 DLP changeable 750 LP BV786 User Guide 127 ...

Page 130: ...8 10 488 10 SSC on 488 493 DLP 493 DLP 525 35 509 24 eGFP FITC 530 DLP changeable 549 15 eYFP 558 DSP 558 DSP 593 52 583 30 PE R phycoerythrin 600 DLP changeable 615 24 PE Texas Red 629 DLP 629 DLP 692 80 692 80 PE Cy5 PerCP Cy5 5 752 DLP changeable 752 DLP changeable 750 LP 750 LP PE Cy7 128 ZE5 Cell Analyzer and Everest Software ...

Page 131: ...een 561 nm 50 mW 7 Detectors Fluorophores 577 15 dTomato dsRed PE 581 DLP changeable 589 15 600 DLP 615 24 PE Texas Red PI bound to dead 629 DLP changeable 640 20 mPlum 652 DLP 670 30 PE Alexa Fluor 647 PE Cy5 690 DLP changeable 720 60 PE Cy5 5 750 DSP 750 LP PE Cy7 User Guide 129 ...

Page 132: ...alyzer system The coated pieces of glass are delicate handle them with care Any scrape or scratch on the surface could significantly affect the light passing through Always wear gloves when removing or replacing filters to avoid depositing smudges and fingerprints on the glass surfaces See Cleaning the Optical Filters on page 334 for specific instructions on cleaning optical filters To replace opt...

Page 133: ...ptical filters by pulling the filter sticks out of their slots in the filter bank as shown in the next figure 4 Insert replacement filter sticks into empty slots as needed Tip Store extra optical filter sticks in the slots in the filter access door User Guide 131 ...

Page 134: ...ers an administrator can use Everest Software to reflect these changes in the optical filter configuration Any changes made to the software configuration will be automatically saved for the current application session To view and edit the optical filter configuration 1 Click Display Optical Filter Configuration in the toolbar The Filter Configuration dialog box opens 132 ZE5 Cell Analyzer and Ever...

Page 135: ...e of wavelengths allowed through the entire detector path 4 Select the label in the final pathway position and replace it with the label for the new filter Tip Notice how the path s allowed wavelength range changes in the spectral plot 5 Do one of the following n To save the changes only for the current application session close the Filter Configuration dialog box n To save the changes for the cur...

Page 136: ...o one of the following n If you selected one of the Copy options paste the graphic into the application of your choice n If you selected the Write option select a location for the file and click Save Reverting to the Default Optical Figure Configuration Administrators can revert to the original filter configuration settings in the software Before performing this task ensure that the original filte...

Page 137: ...ltiple LEDs to pulse ten different wavelengths of light through the instrument s optical detection paths The next figure shows a graph of these light wavelengths The ZE5 EYE runs automatically n As part of the Startup and QC processes n When the optical filter access door is opened for at least 5 sec and then closed User Guide 135 ...

Page 138: ... Before the ZE5 EYE process runs a black circle with a question mark appears next to each detector as shown in the next figure Note If the ZE5 EYE runs while you have plots open in an experiment workspace data might appear in the plots To confirm filter choices using the ZE5 EYE 1 If the ZE5 EYE did not run automatically click the ZE5 EYE button located to the right of the Filter Configuration dia...

Page 139: ...ons ZE5 EYE success or failure is shown as follows n If the ZE5 EYE runs successfully a success confirmation message appears in the status bar and a green check mark appears next to the final filter position for each detector User Guide 137 ...

Page 140: ...nged without a corresponding filter label modification in the software an error appears in the status bar and a red circle with an exclamation point appears next to each position that caused the ZE5 EYE process to fail 138 ZE5 Cell Analyzer and Everest Software ...

Page 141: ...resolve the ZE5 EYE failure ensure that the correct optical filter is placed in its slot and edit the optical filter configuration to reflect this Installing a Neutral Density Filter To alter the range of detection sensitivity of an SSC or fluorescence detector you can replace a bandpass filter with a neutral density filter For more information about neutral density filters see Optical Mirror and ...

Page 142: ...nsity filter preserving its original orientation 6 Re install the metal clip 7 Re insert the filter stick into the instrument Note Installing a neutral density filter causes a reduction in light detection and can cause the affected filter position to be flagged by the ZE5 EYE This ZE5 EYE failure serves as a reminder to remove the neutral density filter at the end of the experiment that requires i...

Page 143: ...eriments and Workspaces n Chapter 8 Acquiring Samples n Chapter 10 Analyzing Saving and Printing Data System Power Do not turn off the ZE5 Cell Analyzer system by using the main power switch The system is safe in standby mode after a software shutdown This facilitates quick or automated startup If you do need to turn instrument power off shut down Everest Software first If the instrument is shut o...

Page 144: ...ining run time with the current bottle levels Ensure that the waste bottles are sufficiently empty and the sheath fluid bottles are sufficiently full but not overfull After logging in you can check fluidics status from the toolbar For more information see the list of fluidics status items in System Tools on page 96 The pair of bottles illuminated by the green LEDs in the bulk fluidics chamber is i...

Page 145: ...ng bottles always wear gloves and minimize air exposure to help avoid contamination n When transferring a cap assembly to a new bottle avoid touching the exterior bottle surface with the cap assembly If you need to set a cap assembly down place it on a sterilized surface n For safety treat all waste as biohazardous User Guide 143 ...

Page 146: ...aste bottles are not changed before they are full the system shuts down to avoid overfilling The sheath DI water bottles are sealed with blue caps Each sheath bottle has a run time of about 4 hr and must be filled when empty Everest Software provides warnings 1 hr 30 min and 5 min before the sheath bottles are nearly empty allowing you to swap the fluidics To avoid running the system dry shutdown ...

Page 147: ...is properly displaced as fluid is pumped into the bottle Any air vented from the waste bottle passes through a 0 2 μm filter The waste fluidics and air vent connections are interchangeable Waste and sheath bottle connections are specifically keyed to their respective ports and cannot be connected to the wrong ports Important When removing the waste bottle ensure that the bottle cap is firmly tight...

Page 148: ...ment Tip An audible click indicates that the tubing is connected Replacing Sheath Bottles Each sheath bottle has a single connection to the instrument Waste and sheath bottle connections are specifically keyed to their respective ports and cannot be connected to the wrong ports Important When refilling the sheath fluid bottles n Ensure that the filter and tubing are not contaminated when you remov...

Page 149: ...s 1 Push the quick connect tabs on the connections to disengage the tubes from the instrument 2 Pull the sheath bottles out of the instrument 3 Unscrew the cap assemblies remove them from the bottles and pull the tubing out of the bottles User Guide 147 ...

Page 150: ...lacing Sheath Additive and Cleaner Bottles The sheath additive and cleaner bottles are located at the bottom of the fluidics chamber They can be refilled or replaced at any time other than during shutdown Sheath additive and cleaner bottle connections are specifically keyed to their respective ports and cannot be connected to the wrong ports the white capped sheath additive bottle always goes on t...

Page 151: ...e bottle out of the instrument 3 Unscrew the cap assembly and remove it from the bottle 4 Refill the white capped bottle with sheath additive refill the blue capped bottle with cleaner or replace a bottle with a new one User Guide 149 ...

Page 152: ...Chapter 6 Daily Routine 5 Carefully place the cap assembly onto the bottle and tighten the cap 6 Place the bottle back into the instrument 150 ZE5 Cell Analyzer and Everest Software ...

Page 153: ...wo panes Instrument Status and User Login as described in Login Window on page 56 If the software is connected to an instrument an instrument picture appears in the Instrument Status pane Before proceeding check the instrument status n A status of Ready indicates that the system has been started up and is running n A status of Off indicates that the instrument has been shut down and is not running...

Page 154: ...er database Starting Up the System Before running samples the instrument must be powered on and Everest Software must perform system startup Bio Rad recommends that you leave the system powered on at all times and that you perform the shutdown procedure in Everest Software at the end of each day Note In Everest Software a system status of Off indicates that the ZE5 Cell Analyzer is shut down but n...

Page 155: ...ll The unclog and ZE5 EYE processes run immediately after the startup process You can run the startup process after logging into the system If your system administrator has allowed it you can also run the startup process before logging into the system For more information see Unclogging the Sample Line and Probe on page 322 Using the ZE5 EYE to Confirm Filter Choices on page 135 Running Quality Co...

Page 156: ...Chapter 6 Daily Routine To start up the instrument 1 Click Startup 2 To view startup details click the down arrow 154 ZE5 Cell Analyzer and Everest Software ...

Page 157: ...e are in the correct starting position and ready to run samples Setting Rinse Pressure Sets cleaner pressure to 12 psi for subsequent steps using cleaner approximately 5 ml of cleaner is used in the startup process Purging Wash Lines Runs deionized water through wash lines Setting Clean Pressure Sets clean pressure to 12 psi for subsequent cleaner steps Getting Sheath Ready Waits for the air pump ...

Page 158: ...eps in the startup process continued After startup the status changes to Ready and the Shutdown button appears in the Login window if you have not logged in yet or in the Home window if you have logged in After logging in run the Quality Control process Running Quality Control The Quality Control QC process can be activated in the following places in Everest Software n Home window 156 ZE5 Cell Ana...

Page 159: ... PMT voltage changes are compared against the QC criteria Finally a 5 000 event file is collected at 150 events per second and voltages and CVs are stored for reporting purposes These values can be displayed based on the date the process was run in daily QC reports and QC trending reports Important While QC is in progress the following functions are disabled n Returning to the Home window n Initia...

Page 160: ... x SSC plot and a univariate histogram for each channel Build Runlist Builds a run list including loader movement accessing the QC bead bottle and bead agitation for the QC process Set Default PMT Voltages Sets PMT voltages to the values determined at the end of the last successful QC process Add Plots Adds plots to the workspace Start Runlist Initializes and begins acquisition Test Validation Eve...

Page 161: ...ur experiment Table 32 Steps in the QC process continued The QC workspace loads automatically regardless of whether it was initiated from the Home window or Acquisition workspace When QC completes the software remains in the Acquisition workspace To return to the Home window click the Home button Note The Home button on the toolbar is disabled during the QC process You can return to the Home windo...

Page 162: ...mples see Chapter 8 Acquiring Samples Pausing the System To conserve bulk fluidics and prolong laser lifetime you can pause the system Important While the system is paused the following functions are disabled n All functions in the Instrument Control PMT Control and Trigger panels n Initiating the ZE5 EYE process n Running the QC process n Shutting down Everest Software and the ZE5 Cell Analyzer n...

Page 163: ... probe and flow cell with cleaner depressurizes the fluidics and logs you out of the system The shutdown process is entirely automated and does not require user interaction Important Always follow the personal protective equipment PPE guidelines relevant to your laboratory s safety procedures for dealing with ethanol or bleach The Shutdown button is located n In the Home window n In the System Act...

Page 164: ...Progress symbol appears If you scheduled an automatic startup the countdown to startup begins Important If Everest Software is not running any scheduled automatic startup will not be performed Leave the software running if an automatic startup is scheduled The sheath bottles must be sufficiently full and the waste bottles must be sufficiently empty to allow 2 hr of run time If the system automatic...

Page 165: ...cludes the instrument s serial number hardware configuration channel and pinhole configuration as well as settings specific to the loader sample pump bulk fluidics fluidics flow rate and volume calibration Setting Rinse Pressure Sets rinse pressure to the configuration value typically 10 or 12 psi for subsequent steps Table 33 Steps in the shutdown process User Guide 163 ...

Page 166: ... down between home and the maximum position through the wash station several times to clean the exterior of the probe with cleaner Probe crash is also checked during this step Purging Wash Lines Purges wash line with DI for 20 sec Rinsing Probe with Sheath Moves the loader to safe position moves the sample probe up and down between home and the maximum position through the wash station several tim...

Page 167: ... and the waste bottles must be sufficiently empty to allow 2 hr of run time After the Auto Startup function is set the countdown to startup begins Important If Everest Software is not running any scheduled automatic startup will not be performed Leave the software running if an automatic startup is scheduled If the system automatically starts up and no user logs in within 1 hr the system shuts dow...

Page 168: ...on or during the startup shutdown decontamination or QC process To exit Everest Software u Click Close in the upper right corner Note If the software cannot exit immediately Bio Rad recommends waiting rather than forcing the software to exit by clicking I Can t Wait 166 ZE5 Cell Analyzer and Everest Software ...

Page 169: ... of the Experiment Builder you assign an experiment name The experiment name also becomes the default name of the first panel in the experiment You can change experiment and panel names on later screens To create a new experiment 1 After logging in click New Experiment on the Home window 2 Enter a name for the experiment and click the Next arrow Tip If needed you can set up a single plate or tube ...

Page 170: ...rophores needed for your experiment To select fluorophores 1 Select the fluorophores to use in the experiment from the list of fluorophores Use the Search box to type at least the first few letters of the fluorophore s name The list narrows to those fluorophores relevant to the search 168 ZE5 Cell Analyzer and Everest Software ...

Page 171: ...hore appear on the emission plot associated with the fluorophore s optimal excitation laser line n The fluorophore is automatically assigned the most appropriate detector based on the instrument s current configuration n The Active checkbox is selected n The Name column is populated with the fluorophore name Tip In a spectral graph containing multiple spectra point to a spectral line to view the n...

Page 172: ...he run list by specifying how samples are processed Settings include media type well tube positions sample names compensation controls volume limit target flow rate or target event rate sampling speed wash agitation temperature addition of reagent and pauses Selecting the Media Type You can select from six predefined media sample input device types or create a custom media type For more informatio...

Page 173: ...Setting Up the Run List The plate setup displays a layout of the selected media User Guide 171 ...

Page 174: ... If you need to change the selected media type click Switch Media in the upper left corner of the plate panel Tip The Experiment Name is the name entered in the Name screen and can be modified here if needed 172 ZE5 Cell Analyzer and Everest Software ...

Page 175: ...predefined media types match the device you intend to use you can create a custom media type To create a custom media type 1 In the Media list click Custom 2 Optional If your custom device is similar to an existing plate device select it to use as a template User Guide 173 ...

Page 176: ...n the Create a New Custom Plate area type a name for the new custom media Important If you select an existing device to use as a template but do not change the Plate Name value the calibration for the existing device will be modified 4 Edit plate settings as necessary and then click Next 174 ZE5 Cell Analyzer and Everest Software ...

Page 177: ...Setting Up the Run List User Guide 175 ...

Page 178: ... that follows indicate the horizontal distance between the centers of two adjacent wells A1 A2 which is 8 73 mm Note If the probe does not drop low enough into a well enter a value for Probe Z Excess Delta to drive the probe down lower If the probe hits the well bottom this information is used in calibrating the probe Z value 6 Edit the dimensions as needed and click Next 7 Place the custom device...

Page 179: ...as needed 9 Determine whether the probe has entered the last position on the custom device n If the probe is centered in the last position click Yes n If the probe is not centered in the last position click No use the arrows to adjust it to the center of the well and click Accept Current Location Repeat this process as needed The layout of the custom device appears in the Plate Setup panel To sele...

Page 180: ...Chapter 7 Creating Experiments and Workspaces The layout of the custom device appears in the Plate Setup panel 178 ZE5 Cell Analyzer and Everest Software ...

Page 181: ...Runs each column in turn starting with column 1 Runs each row in turn starting with row A Runs in serpentine fashion row by row Runs A1 through A8 B8 through B1 C1 through C8 and so forth Runs in serpentine fashion column by column Runs A1 through E1 E2 through A2 A3 through E3 and so forth Allows you to set the sample temperature from 4 37ºC in 1º increments The default is 15ºC The actual tempera...

Page 182: ...lculations Table 34 Plate Settings items and their functions continued Setting the Run List Order You can choose the order in which rows and columns of tubes or wells are sampled To set up the run list order u Click the Run list Order button that displays the order in which tubes or wells should be sampled Note Ensure that any inserted tube rack or plate is oriented correctly with position A1 in t...

Page 183: ... temperature after activating temperature control Specifying Shutdown Upon Completion You can instruct the system to shut down when run list acquisition is complete To specify shutdown upon completion u In the Plate Settings area click the Shutdown When Complete toggle User Guide 181 ...

Page 184: ...e automatically added to the plate map To set up compensation controls 1 In the Template area of the Selected Fluorophores panel select the pulse parameter Area or Height that you want to compensate Note You must select this parameter first the Area and Height buttons are disabled after the compensation template is activated 2 To include a negative control tube or well in the experiment click the ...

Page 185: ...s Manually The Single tab in the Sample Naming panel allows you to label individual positions one at the time You can name positions as needed Names are written to the FCS file associated with each position Note If you turned on Compensation in the Selected Fluorophores panel the compensation template automatically names each Setup position based on the corresponding fluorophore name To label posi...

Page 186: ... label several positions with the same naming components or the whole plate As with single naming the names are written to the FCS file associated with each position For information about the Sample Naming panel see Sample Naming Panel on page 78 Tip You can also label multiple positions at one time by importing the names from a spreadsheet For more information see Importing Names from a Spreadshe...

Page 187: ...e are five available name components n Date n Well ID well position for example A1 B5 n Sequence ID order in which positions will be acquired n Experiment Name n Custom allows you to create your own prefix or suffix 3 Select the checkbox for the components you want to include in the name 4 To use the Custom name component type a value in the Custom box User Guide 185 ...

Page 188: ...ow to apply the names That is whether the name components should n Replace existing names n Be added to the end of existing names append n Be added to the beginning of existing names prepend 6 Do one of the following 186 ZE5 Cell Analyzer and Everest Software ...

Page 189: ...Multiple tab in the Sample Naming panel also allows you to import names from a spreadsheet To assign multiple names to positions using a spreadsheet 1 Create an external spreadsheet with two columns a Populate the first column with position IDs for example B1 B2 B3 corresponding to the samples in the tube rack or plate b Populate the second column with name values 2 In the spreadsheet select the v...

Page 190: ...eriments and Workspaces 5 In the Multiple tab in the Sample Naming panel in Everest Software click Import The names are imported into the corresponding Sample locations in the table 188 ZE5 Cell Analyzer and Everest Software ...

Page 191: ...Setting Up the Run List User Guide 189 ...

Page 192: ...file during acquisition and can be used for batch processing in third party analysis software To set up keywords 1 In the Sample Naming panel click the Keyword tab 2 Type a keyword in the Add New Keyword box and click Add The keyword appears in the Keywords area 3 Repeat Step 2 for additional keywords that you want to add 190 ZE5 Cell Analyzer and Everest Software ...

Page 193: ...e box below the keyword 5 In the plate map select the samples to which to apply a keyword 6 In the Keywords area select the checkbox for each keyword that you want to apply 7 Click Apply to Selected The applied values appear in the Samples area in the Keyword tab User Guide 191 ...

Page 194: ... you do NOT want to remove 2 Select the checkbox for the keyword that you DO want to remove Important Ensure that the checkbox for all other keywords is cleared 3 Click Remove To remove a keyword from a sample position u In the Samples area in the Keyword tab click the x to the right of the keyword box 192 ZE5 Cell Analyzer and Everest Software ...

Page 195: ...l n 96 well deep well plate 1 000 µl n 384 well plate 100 µl Note If you select more than one type of stop limit acquisition for the sample stops when the first stop condition is reached Gate limits are set on the Settings screen of the Experiment Builder If you want to use a gate limit select the Gate checkbox on the Samples screen and then specify the actual gate limit on the Settings screen Was...

Page 196: ...ec The speed and radius of agitation automatically change with the sample media type Tip It might not be necessary to activate agitation for each well For the highest throughput rate it might be necessary to agitate only a few times within the plate Positions in the plate map in which agitation is activated appear as High Throughput Pause After Volumetric Counting and Return Sample Settings By def...

Page 197: ... can be used to verify results achieved or to set compensation values before moving on to the next series of samples Tip Activate Pause After at the last compensation control position so that compensation can be calculated before returning to sample recording The resulting compensation matrix is written to the FCS files Positions in the plate map in which Pause After is activated appear as Volumet...

Page 198: ...ed any sample remaining in the sample line after the required number of events has been acquired is returned to the tube well Flow Rate or Event Rate Settings The flow rate controls allow you to specify a precise target flow rate for each position in µl sec The following flow rate options are available n Low 0 5 µl sec 30 µl min n Normal default 1 0 µl sec 60 µl min n High 1 5 µl sec 90 µl min Not...

Page 199: ... position to enhance mixing Positions in the plate map on which reagent will be added appear as Assigning Position Types Tip This procedure explains how to assign position types for a single panel experiment If needed you can set up a single plate or tube rack with multiple experimental panels For more information see Multipanel Experiments on page 87 and Setting Up Multiple Panels on page 262 Ass...

Page 200: ...appropriate button Selected positions are highlighted with red boxes If you turned on Compensation in the Selected Fluorophores panel Setup positions will have already been configured 2 To change the location of any assigned position a Click the Drag and Drop button in the lower right of the plate map 198 ZE5 Cell Analyzer and Everest Software ...

Page 201: ...gh throughput mode is activated samples are aspirated continuously the sample line contains multiple samples each sample is separated by a boundary of air and wash fluid Select high throughput mode to achieve the highest possible sample throughput For more information about high throughput mode see High Throughput Mode on page 76 To select high throughput mode 1 In the plate map select the positio...

Page 202: ...iles along with the compensation matrix which are exported in the FCS file for analysis in third party software To pause after a tube or well 1 In the plate map select the relevant positions 2 Click the Pause After button The run list pauses after the applicable position has been acquired Positions for which pause after is activated are marked with an image in the plate map For more information se...

Page 203: ...ll If high throughput mode has not been activated you can use the Return Sample function to conserve sample To return sample 1 In the plate map select the relevant positions 2 Click the Return Sample button After the acquisition limit has been reached for the assigned positions the sample pump runs backwards to return any unused sample to its tube or well User Guide 201 ...

Page 204: ...when the total acquisition volume reaches the specified value c Gate sets the volume limit to the maximum for the position You can configure gate limits in the next screen Settings and change the volume to reflect what is in the tube or well if needed 3 To set different limits for other positions repeat Steps 1 and 2 for other positions in the run list Configuring Wash Settings Configure washes as...

Page 205: ...arked with an image in the plate map For more information see Position Images on page 76 Specifying Flow Rate or Event Rate A volumetric target flow rate or target event rate must be assigned for all samples By default a rate of 1 μl sec is assigned when positions are activated See Flow Rate or Event Rate Settings on page 196 for information about the flow rate options Note In a high throughput ru...

Page 206: ...ental reagents see Reagent Settings on page 197 To add reagents to samples 1 Ensure that at least one position in the plate map is designated as a Reagent position 2 Select the sample position to add reagent 3 Click the Reagent button 4 Select the position from which to add reagent in the dropdown list 5 Specify the reagent volume in the Volume box 6 To mix the reagent by aspirating and dispensing...

Page 207: ... image in the plate map For more information see Position Images on page 76 Plate Map in the Instrument Control Panel The plate map in the Acquisition workspace displays real time information on the progress of an experiment It shows the total number of samples programmed to be run If the run has already started the plate map indicates the progress of the run by displaying different colors and sym...

Page 208: ...currently passing through the interrogation point in the flow cell The data presented in the workspace are from this sample position Sample sample from this position has been acquired and an FCS file has been saved Hit detection mode only this position has been acquired and has been classified as a hit Sample hit detection mode only this position has been acquired and an FCS file has been saved po...

Page 209: ... the current panel Reviewing the Run List After you have applied the settings for all of the samples included in the experiment and selected the workspace type review the run list Important After configuring settings in the Plate Setup panel examine the expanded Run List panel to ensure that each sample position has been configured as needed To review the run list and go to experiment settings 1 T...

Page 210: ...ngs are correct 3 Close the Run List Details window and then click the Next arrow to go to the Settings screen In the Settings screen you can create plots for analysis and adjust instrument settings such as thresholds and PMT voltages You can also add experimental panels to a single plate or rack if needed 208 ZE5 Cell Analyzer and Everest Software ...

Page 211: ...he plot builder reflect those that were activated in the Experiment Builder Fluorophores screen You can specify linear log or hyperlog scaling for each axis specify the pulse measurement area height or width and enable display of compensated data as needed For more information see Creating Density Plots on page 211 Creating Histograms on page 212 and Creating Time Plots on page 213 Plots Created b...

Page 212: ... n A histogram for the control s channel that includes a region for identifying the positive population n A plot for each other fluorophore in the experiment so that spillover of the position s fluorophore into each other detector can be assessed and corrected Each fluorescence density plot in a compensation control workspace is configured with the current fluorophore s detector on the x axis and ...

Page 213: ...e Log button is blue click it so that it is no longer blue n For log scale if the Log button is blue leave it selected if the Log button is not blue click it n For hyperlog scale leave Log selected and click HyperLog so that both the Log and HyperLog buttons are blue 4 Specify whether to display compensated data for the parameter 5 Select the pulse measurement height area or width for the paramete...

Page 214: ...e if the Log button is blue click it so that it is no longer blue n For log scale if the Log button is blue leave it selected if the Log button is not blue click it n For hyperlog scale leave Log selected and click HyperLog so that both the Log and HyperLog buttons are blue 4 Specify whether to display compensated data for the parameter 5 Select the pulse measurement height area or width for the p...

Page 215: ...t so that it is no longer blue n For log scale if the Log button is blue leave it selected if the Log button is not blue click it 4 Select the pulse measurement height area or width for the y axis 5 Select a time range for the x axis Time range option Description Sliding As time progresses display range remains the same but slides to reflect current time Continuous As time progresses time range in...

Page 216: ...ify whether histogram data are displayed in linear log or hyperlog scale and specify the pulse measurement area height or width Tip Histograms are created only for parameters that are activated in the workspace Checkboxes next to each laser allow you to specify histogram creation for a particular detection path To create histograms for all channels 1 Click Advanced Plot Builder in the toolbar The ...

Page 217: ...only if you have set filters or regions on plots that you previously created in the current experiment 4 Specify linear or log scale Note The default scale is linear although Log appears to be selected To specify log scale click Log until the box appears blue For example User Guide 215 ...

Page 218: ...Chapter 7 Creating Experiments and Workspaces 5 Select the pulse measurement height area or width for the parameter The histograms are added to the workspace 216 ZE5 Cell Analyzer and Everest Software ...

Page 219: ...s only to density and time plots Adding Regions to Density Plots and Time Plots on page 225 Add Rectangle Region adds a rectangle region to the plot Applies only to density and time plots Adding Regions to Density Plots and Time Plots on page 225 Add Ellipse Region adds an ellipse region to the plot Applies only to density and time plots Adding Regions to Density Plots and Time Plots on page 225 A...

Page 220: ...egion allows you to add regions to a heat map Applying Heat Maps on page 244 Assign Gate Limit Regions allows you to assign a gate limit to a region so that acquisition stops when the event count in the region reaches the specified limit Applying Filters Gates on page 236 Add Diagonal Separation adds a diagonal line to the plot This graphical element can aid you in setting PMT voltages for compens...

Page 221: ...6 Remove Filter removes a gate assignment from the plot Applying Filters Gates on page 236 Export to PNG saves a PNG of the plot to a location that you specify Exporting Plots and Histograms on page 256 Add Annotation allows you to add a note to the plot Adding Annotations to Plots on page 251 Table 37 Plot and histogram tools and their functions continued Modifying Plot Parameters For each axis o...

Page 222: ...ted data d Select the pulse parameter height width or area e Enter the number of log decades to display f Specify 256 x 256 or 512 x 512 plot resolution bin g Enter the number of hyperlog negative decades to display h Use the slider to select a hyperlog linear coefficient 4 Click Apply to save the changes Important Clicking the X in the upper right corner of the Edit Plot dialog box removes the pl...

Page 223: ... To manage plot statistics 1 Point to a plot or histogram for which you want to manage statistics The plot and histogram toolbar appears 2 Click Manage Statistics 3 In the Statistics dialog box select the items you want to view and clear the items you want to hide 4 Point outside the plot to save your changes User Guide 221 ...

Page 224: ...nces For more information see Specifying Statistics Preferences on page 119 If you do not see the statistics that you want to display contact your system administrator Note If you plan to add any regions to a plot or histogram add them before viewing statistics To view plot statistics 1 On the plot click the Statistics down arrow The plot and region s statistics appear in a table within the plot 2...

Page 225: ...s can be viewed in real time as acquisition occurs Use this feature after applying an experiment to a workspace To compare statistics 1 In the Tools area of the toolbar click Add Statistics A statistics window appears in the workspace 2 Point to the statistics window The Select Statistics and Select Gates toolbar buttons appear 3 Click Select Statistics 4 In the Statistics dialog box select the it...

Page 226: ...filters gates for which you want to compare statistics 7 Point outside the Statistics window to save your changes The statistics for the selected gates appear along with comparisons to all events in the gated plot The gating hierarchy is shown and for gates on density plots values for the x axis and y axis are shown 224 ZE5 Cell Analyzer and Everest Software ...

Page 227: ...s u Point to the plot The plot and histogram toolbar appears To add a rectangle region to a plot 1 In the plot and histogram toolbar click Add Rectangle Region A rectangle is added to the plot 2 To move the rectangle to another part of the plot point inside the region When the pointer changes to a hand use it to drag the rectangle 3 To resize the rectangle point to its outline User Guide 225 ...

Page 228: ...Chapter 7 Creating Experiments and Workspaces When the pointer changes to a double headed arrow use it to drag an edge of the rectangle 226 ZE5 Cell Analyzer and Everest Software ...

Page 229: ...lot and histogram toolbar click Add Ellipse Region An ellipse is added to the plot 2 To move the ellipse to another part of the plot point inside the region When the pointer changes to a hand use it to drag the ellipse 3 To resize the ellipse point to its outline User Guide 227 ...

Page 230: ...en the pointer changes to a hollow plus sign use it to drag an edge of the ellipse 4 To rotate the ellipse point inside the region When the rotation arrow appears on one corner drag it to rotate the ellipse 228 ZE5 Cell Analyzer and Everest Software ...

Page 231: ... click Add Quadrant Regions Quadrants are added to the plot 2 To move the quadrant dividers point to the center circle When the pointer changes to a hand use it to drag the horizontal divider up or down or use it to drag the vertical divider to the left or right 3 To skew the quadrant dividers point to a circle along a plot axis User Guide 229 ...

Page 232: ...ircle along the axis This changes the shape of two of the quadrants at the same time Note Do not apply hit detection data tracking or heat maps to quadrant regions in the Settings workpace wait until you have applied the experiment to the Acquisition workspace 230 ZE5 Cell Analyzer and Everest Software ...

Page 233: ...click Add Polygon Region 2 Click inside the plot where you want the first corner of the polygon 3 Move the pointer and click again to create more corners of the polygon 4 Double click to finish drawing the polygon 5 To move the polygon to another part of the plot point inside the region User Guide 231 ...

Page 234: ...ter changes to a hand use it to drag the polygon 6 To rotate the polygon point inside the region When the rotation arrow appears on one corner drag it to rotate the polygon 7 To resize the polygon point to its outline 232 ZE5 Cell Analyzer and Everest Software ...

Page 235: ...istogram Tools When the pointer changes to a double headed arrow use it to drag an edge of the polygon To delete any type of region from a plot u Click the x that appears on the upper right of the region User Guide 233 ...

Page 236: ...Point to the histogram The plot and histogram toolbar appears 2 In the plot and histogram toolbar click Add Bar Region A bar region is added to the histogram 3 To move the bar region to the left or right point inside the region When the pointer changes to a four headed arrow use it to drag the bar region 4 To resize the bar region point to its left or right edge 234 ZE5 Cell Analyzer and Everest S...

Page 237: ...Renaming Regions You can rename regions created in plots or histograms Bio Rad recommends that you rename regions before you base gates on the regions To rename a region 1 Double click the region name The region name is selected 2 Type new name and press Enter Tip If a region name overlaps another plot feature such as a rotation arrow or gate limit symbol you can drag the region name to move it Us...

Page 238: ...de the rest Note When you create a rectangle ellipse or polygon region Everest Software creates a NOT gate that includes events that fall outside of the region To apply a filter to a plot or histogram 1 Point to the plot or histogram that you want to filter The plot and histogram toolbar appears 2 Click Apply Filter A list of available filters appears 3 Select the checkbox for the filter that you ...

Page 239: ...y the data that fall into the region applied in the filter To remove a filter from a plot or histogram 1 Point to the plot or histogram 2 Click Remove Filter in the plot and histogram toolbar that appears The filter is removed form the plot or histogram User Guide 237 ...

Page 240: ...entially you can create a filter gate that uses multiple regions To apply sequential filters to a plot or histogram 1 Create a region on a plot or histogram 2 Point to a second plot and click Apply Filter in the plot and histogram toolbar A list of available filters appears 238 ZE5 Cell Analyzer and Everest Software ...

Page 241: ... Plot and Histogram Tools 3 Select the region that you created in the first plot The applied region is shown in the upper left corner of the plot or histogram 4 Add a region to the second plot User Guide 239 ...

Page 242: ...n the pop up toolbar 6 Select the region that you created in the second plot The filter in the upper left corner of the plot or histogram indicates that the plot shows the subset of data from the first region R3 that also appear in the second region R4 240 ZE5 Cell Analyzer and Everest Software ...

Page 243: ...or histogram that contains the region s that you want to gate The plot and histogram toolbar appears 2 Click Assign Gate Limit Regions 3 In the Available Gate Limit Regions area that pops up select the checkbox for each region that you want to gate 4 If needed change the Event Limit value for each gated region 5 Point outside the plot or histogram to save your changes A gate symbol appears to the ...

Page 244: ...lots for the Experimental Sample on page 366 Assigning Data Track Regions You can use data track regions to monitor for clogs or sample disturbances during acquisition When you assign data tracking to a region in a density plot you specify a target percentage During acquisition if the percentage of events drops below the target acquisition pauses and you are notified so that you can determine what...

Page 245: ...in of error to avoid pausing acquisition for samples with regions that come very close to meeting the target percentage To remove data tracking from a region 1 Point to a density plot that contains the region that uses data tracking The plot and histogram toolbar appears 2 Click Assign Data Track Regions 3 Select None 4 Point outside the plot to save your changes The region outline reverts to yell...

Page 246: ...eat map represented as a pie chart Colors and color gradients provide visual indicators of relative richness The color intensity of a region s pie slice corresponds to the percentage of the sample s events coming from that region n Low percentages appear lighter and gray n High percentages appear darker and red Note When applying heat maps to quadrant regions wait until the experiment has been app...

Page 247: ... Plot and Histogram Tools 2 Click Assign Heat Map Region 3 In the Available Heat Map Regions dialog box that appears select the checkbox for each region that you want to apply to the heat map User Guide 245 ...

Page 248: ...r 7 Creating Experiments and Workspaces 4 Point outside the plot or histogram to save your changes Heat mapping is indicated with a pie chart symbol above the region 246 ZE5 Cell Analyzer and Everest Software ...

Page 249: ...gion that you want to remove from the heat map 4 Point outside the plot or histogram to save your changes The pie chart symbol is removed from the region and the region is no longer applied to the heat map Configuring Hit Detection In hit detection mode Everest Software provides real time feedback regarding whether a population is present or absent in a given region for each tube or well You can u...

Page 250: ...ant to use hit detection The plot and histogram toolbar appears 2 Click Assign Hit Regions 3 In the Available Hit Region dialog box select the checkbox for the region where you want to use hit detection 4 If needed modify the threshold percentage for hit detection in the Threshold box The default value is 15 The hit region is indicated with a target symbol 5 Point outside the plot or histogram to ...

Page 251: ...in setting PMT voltages for a compensation control In a typical scenario the parameter to be compensated is shown on the x axis as in the plots that Everest Software creates automatically for compensation controls If a positive population falls below the diagonal line this indicates that the control sample is brighter in the intended channel than in the channel shown on the y axis Thus the require...

Page 252: ...ts or histograms n After setting up a rectangle ellipse or polygon region on a plot you can copy this region to all other plots in the workspace n After setting up a bar region on a histogram you can copy this region to all other histograms in the workspace n After setting up a region on a plot or histogram you can apply this region as a filter on all other plots or histograms in the workspace Not...

Page 253: ...ilter to apply the region as a filter on all other plots 4 Repeat Step 3 for other regions as needed 5 Point outside the plot or histogram to save your changes Adding Annotations to Plots You can annotate a plot or histogram directly To add an annotation to a plot or histogram 1 Point to the plot or histogram that you want to annotate The plot and histogram toolbar appears 2 Click Add Annotation A...

Page 254: ...ce it with your annotation 4 To change the font size double click the text and click the T symbol in the toolbar that appear Use the slider to increase or decrease the size 5 To move the annotation drag it to another part of the plot or histogram 252 ZE5 Cell Analyzer and Everest Software ...

Page 255: ... based assays For more information about time plots see Creating Time Plots on page 213 To use a ratio in a time plot 1 Decide which parameter you want to use as the numerator in the ratio and which parameter you want to use as the denominator 2 Click Create Time Plot in the Settings window toolbar 3 Specify axis scaling display of compensated data pulse measurement and time range as needed 4 Clic...

Page 256: ...eriments and Workspaces 6 Select a second parameter to use for the ratio denominator 7 Specify axis scaling display of compensated data and pulse measurement as needed 8 Click Apply 254 ZE5 Cell Analyzer and Everest Software ...

Page 257: ...Using Plot and Histogram Tools The modified time plot appears The y axis uses the ratio of the first parameter to the second parameter User Guide 255 ...

Page 258: ...lot as a graphic 1 Point to the plot or histogram that you want to export The plot and histogram toolbar appears 2 Click Export to PNG 3 In the Save As dialog box browse to a location in which to save the file 4 Type a name for the file and click Save The plot or histogram is exported as a PNG file 256 ZE5 Cell Analyzer and Everest Software ...

Page 259: ...e the Settings screen of the Experiment Builder to enter initial instrument settings such as PMT voltages laser power and primary and secondary trigger and threshold Note The default PMT voltage values are those set at the most recent QC baseline The default threshold value for both FSC and SSC triggers is 10 User Guide 257 ...

Page 260: ...to the flow cell Note The shutter must be open to allow parameter selection from that laser The shutter automatically closes if no parameters for the laser have been selected in the Experiment Builder Edit laser power allows you to specify power settings for individual lasers in steps from 10 mW to the maximum power output Neutral density checkboxes if selected a 2 0 ND filter is moved in front of...

Page 261: ...e values are included in the FCS file Trigger use the trigger parameter or parameters to alert the system to the presence of an event over the threshold The trigger plot represents what the electronics are detecting it uses log scaling on the x axis and data in the gray region are excluded Raising or lowering the threshold allows you to exclude unwanted data from acquisition Data below the thresho...

Page 262: ... to 100 The default value is 10 00 Important The threshold adjustments are saved only for the current experiment The default threshold value for the primary trigger which is enabled for each new experiment is 10 The default threshold value for the secondary trigger which is disabled for each new experiment is 10 when the trigger is enabled The adjusted threshold values are included in the FCS file...

Page 263: ...position is indicated by the dotted line Although the threshold plot shows every event measured in this detector events below the threshold are not saved in the data file If two triggers are selected the primary trigger threshold is indicated by the vertical dotted line and the secondary trigger threshold is indicated by the horizontal dotted line Table 38 PMT and Laser Control Items continued Use...

Page 264: ...he Settings screen For more information about multipanel experiments see Multipanel Experiments on page 87 To set up a multiple panels 1 After defining the first panel click Panel in the bottom navigation bar in the Settings screen 2 In the Name screen type name for the new panel in the Current Panel box 3 Click the Next arrow to go to the Fluorophores screen 4 Define a new set of activated parame...

Page 265: ...ng Up Multiple Panels 5 Click the Next arrow to go to the Samples screen Positions programmed for the previous panel are shown in black any positions created for the new panel appear as usual User Guide 263 ...

Page 266: ...ons and create gates for the new panel 9 Click Apply to apply the experiment including all of the panels to the workspace When you move to Acquisition mode the ZE5 Cell Analyzer will acquire the whole plate or set of tubes in a single run and will respect the settings adjustments optimized for each panel in Setup mode there is no need to reoptimize settings between the panel runs To adjust setting...

Page 267: ...trument settings in the Settings screen of the Experiment Builder you are ready to acquire your samples as explained in Chapter 8 Acquiring Samples To run or save the experiment u Do one of the following n To load the completed run list click Apply n To save the run list for future use click Export User Guide 265 ...

Page 268: ...eate plots add regions apply filters and adjust settings Sample acquisition and data recording are available directly from this screen When you run a stat tube all parameters are activated by default and can be named by editing the name in the parameter list See the following sections for more information n Chapter 8 Acquiring Samples n PMT and Laser Controls on page 258 n Creating Plots and Histo...

Page 269: ... experiment that is based on a plate or tube rack An added stat tube can be assigned only the Sample position type it cannot be assigned as Wash Reagent or Setup Agitation temperature and high throughput settings are not applicable to stat tubes To add a stat tube to an experiment 1 In the Plate Setup pane click Add Stat Tube User Guide 267 ...

Page 270: ...r the stat tube 3 Click Sample to select the Sample position type for the stat tube 4 Specify other settings for the stat tube such as stop limits and target event or flow rate 5 Click Plate View to return to setting up the plate or rack 268 ZE5 Cell Analyzer and Everest Software ...

Page 271: ...Running a Quick Stat Sample The added stat tube is indicated with a symbol in the upper left area of the plate map User Guide 269 ...

Page 272: ...it might appear in the list If it is not recent you can browse to find it To run an existing experiment 1 Return to the Home window 2 In the Recent Experiment Sessions panel expand the list of recent experiments 3 Do one of the following n If the experiment session that you want to run is in the list of recently displayed items click Run for the experiment session The experiment session is applied...

Page 273: ...lick Load Run List in the upper right b Browse to find the experiment that you want to run c Select the rlst file and click Open The experiment opens in the Experiment Builder where you can make any needed adjustments before applying it to the workspace For more information see Recent Experiment Sessions on page 62 User Guide 271 ...

Page 274: ...trix n Plots Sample positions are not imported This allows you to apply the settings to different media and different configurations of samples To import settings u Do one of the following n In the Home window click Import Settings Find the run list file that contains the desired settings select it and click Open n In the Home window expand the list in the Recent Experiment Sessions panel For the ...

Page 275: ...screen you can use the imported fluorophore and detection settings as is or you can modify them In the Samples screen you will need to select a media type and set up controls and samples for the experiment because these settings are not imported If you used the compensation template in the previous experiment the same compensation template will be applied User Guide 273 ...

Page 276: ...Chapter 7 Creating Experiments and Workspaces 274 ZE5 Cell Analyzer and Everest Software ...

Page 277: ...applying it load your samples into the instrument To load a sample 1 Press the silver sample chamber button on the front of the instrument to extend the loader 2 Install a tube rack plate or stat tube on the loader in the appropriate position depending on the workspace that has been created for the experiment Note Ensure that any inserted tube rack or plate is oriented correctly with position A1 i...

Page 278: ...p mode you can adjust the threshold and PMT voltages and create gates You can also use the Record function to manually save data to files This gives you more control over when data are saved during the run Data are only saved after you click Record Recording stops after the stop conditions have been met or when you manually stop recording For a stat tube the Setup button does not appear sampling o...

Page 279: ...ta to an FCS file Play moves the probe to the selected position lowers it and turns on the sample pump to run sample Begins streaming data to Everest Software Stop ends data acquisition and turns off the sample pump Replaces the Play button when sampling begins Pause Run List pauses sampling Time gaps that correspond to pause times appear in the data file Resume Run List resumes sample flow Replac...

Page 280: ...click the corresponding button Change the value in the box by clicking the plus and minus buttons for volume rate only or by entering it directly Tip 0 1 µl is the smallest flow rate Table 39 Setup Mode Control Items continued Acquiring Initial Sample in Setup Mode Before proceeding through these steps ensure that you have set up an experiment using the Everest Experiment Builder unless you are ru...

Page 281: ...re enables these functions when sample acquisition is complete To acquire sample in Setup mode 1 Ensure that the correct experiment has been created and loaded 2 Load the sample 3 Click Setup in the Instrument Control panel Note The workspace status bar displays the current mode For a single stat tube the Setup button does not appear sampling occurs in Setup mode by default User Guide 279 ...

Page 282: ...er 8 Acquiring Samples 4 In the plate map click the position from which to start sampling The current position is outlined in red 5 Click Play to initiate sampling 280 ZE5 Cell Analyzer and Everest Software ...

Page 283: ...irst limit is reached a Adjust limits as needed b Click Record Data are collected until the specified limit is reached To stop acquisition click Stop c After data have been recorded for a tube or well the position in the plate map is indicated by a black check mark For information about symbols shown in the plate map see Plate Map in the Instrument Control Panel on page 205 9 After the first posit...

Page 284: ...at is currently in progress Cycle Mode when activated plots display only a certain number of events based on time period specified in this box After the interval is reached data are cleared from the plots and newly acquired events appear Event Rate and Volume during sampling displays the triggered event rate the event count and the volume that has been acquired from the sample during the current r...

Page 285: ... Experiment Builder Note Data are not acquired from any stat tube that has been added on to the run list for a plate or tube rack Important While the system is acquiring sample in Acquisition mode the following functions are disabled n Initiating the ZE5 EYE process n Shutting down Everest Software and the ZE5 Cell Analyzer n In the Instrument tools o Returning sample probe to home position o Clea...

Page 286: ... a position has been sampled its position on the map is indicated by a black check mark For information about symbols shown in the plate map see Plate Map in the Instrument Control Panel on page 205 Tip By default sampling begins at position A1 in the plate map but you can begin acquisition from any sample position Click a position in the plate map and click Start Run List The run list proceeds as...

Page 287: ... setup and sample type by acquiring control positions in Setup mode as described in Acquiring Initial Sample in Setup Mode on page 278 2 Click Acquisition in the Instrument Control panel 3 Click Start Run List Acquisition progress is indicated on the positions on the plate For information about symbols shown in the plate map see Plate Map in the Instrument Control Panel on page 205 Note Because th...

Page 288: ...for example then resume to complete the data file To pause sample acquisition u In the Instrument Control pane in Setup mode click Pause Run List To resume sample acquisition after a pause u In the Instrument Control panel in Setup mode click Resume Run List Stopping Sample Acquisition Stopping sample acquisition stops the sample pump The probe exits the sample and is washed if wash has not been d...

Page 289: ... run list as it was last acquired into the workspace If files were acquired for a portion of the experiment these files appear in the plate map and are exported along with any new files acquired To stop an experiment 1 In the Instrument Control panel click Stop Run List 2 Allow acquisition of the sample to finish 3 In the toolbar click Home To resume an experiment that you just stopped 1 In the Ho...

Page 290: ...ting the full experiment to a compressed ZIP file the software labels the file with only the username and experiment name For example D EverestUsers Export vbala 5Color_2 zip Important Exported data that do not include the full experiment including the run list telemetry and all FCS files do not appear in the Recent Experiments panel on the Home window Exporting the FCS Data File for a Single Posi...

Page 291: ...ingle position click the export button for the position in the right column 3 In the Browse For Folder dialog box that appears do one of the following n Click OK to accept the default location D EverestUsers Export n Click Make New Folder to create a new folder in the Export folder or browse to another location rename the new folder and Click OK Everest Software saves the FCS file for that positio...

Page 292: ...ed position and compress to ZIP n Run List and FCS Files exports run list to RLST format and export all FCS files for the selected positions n Full Experiment to Zip exports full experiment including run list telemetry and all FCS files for the full experiment including all sessions and compress to ZIP 4 By default each export option includes the FSC SSC and fluororphore information including the ...

Page 293: ...tware permits visualization of both compensated and uncompensated data in the same workspace Be sure to select the Comp parameter for plot axes so that the compensation matrix is applied to the data For information on applying compensation to multicolor experiments see Appendix B Example 9 Color Immunophenotyping Experiment Setting Up the Experiment Before applying compensation in Everest Software...

Page 294: ...es panel enable the Negative Control if your experiment includes one 4 Enable Compensation to automatically set up fluorophores as controls 5 To use the negative control as a universal negative in automatic compensation calculations select the Use Universal Negative checkbox 292 ZE5 Cell Analyzer and Everest Software ...

Page 295: ...Setting Up the Experiment 6 Set up samples User Guide 293 ...

Page 296: ...n see High Throughput Pause After Volumetric Counting and Return Sample Settings on page 194 7 Proceed to the workspace Settings screen Everest Software sets up a unique workspace including plots for each compensation control Tip When creating plots for the samples select the Comp option for each axis in the plot builder to show compensated parameters on the plots 8 Click Apply to apply the experi...

Page 297: ...Compensation template record all of the control positions either by using the Record button in Setup mode or running the control samples in Acquisition mode Tip To export the relevant compensation matrix with each experimental FCS data file ensure that compensation has been calculated before running experimental samples 2 After acquiring controls click Analyze in the Quick Actions area of the tool...

Page 298: ...e 4 Repeat step 3 for all the control positions adjusting the gate in the parameter s histogram if needed 5 Click the down arrow next to the Compensation button 6 Select options as needed n Prevent Automatic Region Determination If this option is not selected Everest Software performs automatic region determination and adjusts the regions in all plots for each control 296 ZE5 Cell Analyzer and Eve...

Page 299: ...ncludes controls found to be invalid according to the algorithms that segment the positive population from the negative population If this option is not selected Everest Software excludes them Excluding invalid controls might be necessary if there was no clear separation between positive and negative populations or if a sufficient number of events could not be obtained for a particular single colo...

Page 300: ...rest software offers two options to manually adjust compensation n Dragging populations n Editing the compensation matrix directly Note Unless you know how to perform statistically correct compensation adjustments for all fluorophores used in your experiments Bio Radrecommends that you use automatic compensation Dragging Populations You can adjust compensation by dragging populations in plots eith...

Page 301: ...e region instead of the population Ensure that the hand is outside of a region before dragging it 3 Repeat Step 1 and Step 2 for each single stained control Editing the Compensation Matrix You can adjust compensation by editing the compensation matrix either in the Acquisition tab or in the Analysis tab Note If you plan to perform manual compensation you should understand how to match medians to e...

Page 302: ...eives spillover signal from other detectors Cells in the matrix are shaded green yellow and red to indicate increasing percentage values 4 Save the adjusted compensation values by clicking the X in the upper right corner Tip When viewing compensated data ensure that the Comp option has been selected for the plot axes otherwise non compensated data are displayed 300 ZE5 Cell Analyzer and Everest So...

Page 303: ...LST files FCS files are formatted using the 3 1 standard and thus can be analyzed using compatible third party applications such as FlowJo WinList and Kaluza Run list RLST files are specific to Everest Software They contain all of the information related to an experiment including fluorophores samples sampling settings instrument settings plots and the compensation matrix Run list files are used t...

Page 304: ... the session included sample acquisition with data recording It contains an FCS file for each position Analyzing Data After running an experiment in the Acquisition tab you can analyze it in the Analysis tab Note Wait for acquisition to complete or stop acquisition before moving data to analysis To access the Analysis tab 1 Acquire data in the Acquisition tab 2 Do one of the following n Click Anal...

Page 305: ...eriments To load an experiment in Analysis 1 Click the Analysis tab 2 In the Recent Experiment Sessions panel expand the list of recent experiments 3 Do one of the following n If the experiment session that you want to analyze is in the list of recently displayed items double click it User Guide 303 ...

Page 306: ...orkspace Note For either method the data FCS files are stored separately from the RLST file so those must be present within the folder as well 4 Click a sample position in the plate map to view data specific to that sample Resuming Experiment Analysis During analysis you can click Home to return to the Analysis start window From here you can either click Resume to resume analysis of the previous e...

Page 307: ...ating Histograms on page 212 Create Time Plot creates a plot of time x axis versus a selected parameter y axis For more information see Creating Time Plots on page 213 Add statistics opens a statistics window in it you can select the plot statistics to display for a particular filter gate such as concentration count CV percent of total maximum mean median minimum mode percent of plot standard devi...

Page 308: ...l Instrument send updated run list back to instrument for acquisition for example after compensation is applied Table 41 Analysis toolbar buttons and their functions continued Working with Plots and Statistics in the Analysis Tab In the Analysis tab you can continue to modify experiment plots and statistics using the same controls that were available on the Acquisition tab For more information see...

Page 309: ...s for multiple sample positions for presentation purposes make these modifications in the Analysis tab then use the Batch Print feature of the Publish tab to save the report to PDF or print it For more information see Printing a Report for All Positions on page 312 Working with Compensation in the Analysis Tab In the Analysis tab you can either view the compensation matrix and edit it manually or ...

Page 310: ...a to CSV 1 Click Export CSV in the Share section of the toolbar 2 Select the checkbox for each statistic that you want to include 3 Click Export 4 In the Save As dialog box click Save The data are saved to a CSV file For information on exporting data to FCS and RLST files see Exporting FCS and RLST Data Files on page 288 308 ZE5 Cell Analyzer and Everest Software ...

Page 311: ...sition tab 2 Acquire the samples again Publishing Data After analyzing experiment data in the Analysis tab you can create and print reports in the Publish tab To access the Publish tab 1 Select a sample position in the plate map on the Analysis tab 2 Do one of the following n Click Move to Publish in the Share section of the toolbar n Click the Publish tab The report for the selected position open...

Page 312: ... for a selected plot Note This control appears after you select a plot Position provides x and y coordinates to assist with alignment of plots as you move them Delete selected plot removes the selected plot from the report Table 42 Publish toolbar buttons and their functions Preparing Reports In the Publish tab you can prepare a detailed report for a selected sample position by rearranging or dele...

Page 313: ...lected Position You can either send a report for one position directly to a printer or save it as a PDF for later printing or viewing To print a report for one position 1 Select the position in the plate map of the Publish tab 2 In the Publish toolbar click Print 3 Do one of the following n To send the report to a printer a Select a printer on your network b Click Print n To generate a PDF for the...

Page 314: ...ng or viewing To print a report for all positions 1 In the Publish toolbar click Batch Print Everest Software prepares the report and displays a status bar near the bottom of the screen 2 Do one of the following n To send the report to a printer a Select a printer on your network b Click Print n To generate a PDF for the report a Select Adobe PDF b Click Print c In the Save PDF As dialog box brows...

Page 315: ...ave administrative rights Button Function QC Report opens the most recent daily QC report You can also view reports from previous dates and times Available to all logged in users QC Trending Report opens the QC Trending report You can specify a date range for data display Available to logged in users who have administrative rights ZE5 EYE Trending Report opens the EYE Trending report You can speci...

Page 316: ...e QC status is Passed A red x appears next to those detectors whose values do not pass the QC criteria and the software displays the reason for the failure in the Note column In this case the QC status is Failed for example To view a QC report from a previous date 1 In the Reports section of the toolbar click QC Report 2 Click the calendar button to select a previous date 314 ZE5 Cell Analyzer and...

Page 317: ...date click the time selector and select a time To perform other actions on QC reports u Point to the report and select an option from the buttons that appear Button Function Export the report to CSV Move the data file to analysis Print the report Save the report User Guide 315 ...

Page 318: ...style solid versus dotted To access a QC trending report 1 In the Reports section of the toolbar click QC Trending Report 2 Set the date range for the report by selecting dates from the Start date and End date calendars 3 Select the parameters for which you want to view trends 4 To view a QC trending report for a different laser click its tab 5 To export the QC trending report to a comma delimited...

Page 319: ...e 45 and Using the ZE5 EYE to Confirm Filter Choices on page 135 To access the EYE trending report 1 In the Reports section of the toolbar click ZE5 EYE Trending Report 2 Set the date range for the report by selecting dates from the Start date and End date calendars 3 Select the channels for which you want to view trends 4 To view a ZE5 EYE trending report for a different laser click its tab 5 To ...

Page 320: ...e reports can be printed or exported for reference The User Reports button is available in the Home window when a person with an Admin account is logged in to Everest Software To generate a user report 1 In the Home window click User Reports 2 Set the date range for the report by selecting dates from the Start date and End date calendars 3 To export the report to a CSV click Export Data 318 ZE5 Ce...

Page 321: ...ith the onboard cleaner If additional cleaning is needed it can be run prior to the shutdown process See Cleaning the Sample Line and Probe on page 324 See Cleaning Solutions on page 321 for details regarding approved cleaners for the system tubing To perform the system shutdown procedure 1 Ensure that the onboard cleaner bottle contains sufficient fluid 2 Click Shutdown in the Home window to init...

Page 322: ...nnual preventative maintenance PM plan offered with the ZE5 Cell Analyzer The PM plan includes but is not limited to an annual onsite visit by a Bio Rad Service engineer to n Replace peristaltic pump heads 6 n Replace the sample cartridge n Clean the overflow sensor n Replace the sample probe n Replace the sheath cleaner and additive filter cartridges 3 n Replace the bulk fluid filters 4 n Replace...

Page 323: ...solution further depending on the pathogenicity of the sample Disinfectants for Use in Sheath Line Use a bleach solution containing 580 ppm active chlorine a 1 100 dilution which is roughly equivalent to 1 active chlorine This solution can be used for running the Decontamination wizard See Decontaminating the System on page 325 Disinfectants for Use in the Waste Bottles Use a 1 10 bleach dilution ...

Page 324: ...stem cleaner and DI water This process moves the probe to the port behind the stat tube and cycles through the unclog process To unclog the sample line and probe 1 In the System section of the toolbar click Unclog Sample Line in the Instrument Tools dropdown list 2 To view unclog details click the down arrow 322 ZE5 Cell Analyzer and Everest Software ...

Page 325: ...this process is complete but you can choose to run the QC process to confirm that the clog has been cleared Important To avoid clogs when working with cells resuspend cells into single cell suspension and use a 40 µm or smaller filter prior to cell analysis User Guide 323 ...

Page 326: ...be of ethanol or bleach solution See Cleaning Solutions on page 321 for recommended cleaning solutions to use in the sample line To clean the sample line and probe 1 In the System section of the toolbar click Clean the Sample Probe in the Instrument Tools dropdown list 2 Follow the directions that appear on screen The system is ready for use when this process is complete but you can choose to run ...

Page 327: ...ics can also contribute to contamination despite having internal filters built into the lines To test for contamination disconnect the waste bottle cap collect fluid in the waste input line and culture it The wizard guides you through the necessary steps to complete the decontamination process which takes about 4 hr When the process completes the system is ready to run samples although you can run...

Page 328: ...leach filtered through a 0 2 µl filter flask or syringe filter Preparing for the Decontamination Process Important When handling sheath fluid and DI water bottles always wear gloves and minimize air exposure to help avoid contamination Before beginning the decontamination process you must n Decontaminate the outside of the probe with 10 bleach solution n Prepare bleach and rinse bottles for decont...

Page 329: ...tion 5 Run the stat tube acquisition with the filtered bleach solution for 5 min at 1 µl sec 6 After 5 min stop the stat tube acquisition and remove the stat tube 7 Insert a stat tube containing 4 0 ml of DI water into the stat tube position 8 Run the stat tube acquisition with DI water for 5 min at 1 µl sec to remove any bleach from the outside of the probe To prepare the bleach bottles for decon...

Page 330: ...nating the System Important The decontamination process takes approximately 4 hr to complete During the process you will be prompted to n Replace both sheath bottles approximately 50 min into the procedure with sheath bottles filled with 2 5 L of DI water n Replace the additive and cleaner bottles filled with 200 ml of DI water n Replace both sheath bottles again approximately 190 min into the pro...

Page 331: ...have performed the steps in Preparing for the Decontamination Process on page 326 The Decontamination wizard will prompt you to replace the existing bottles 2 In the System section of the toolbar click Decontamination in the Instrument Tools dropdown list The Decontamination wizard starts User Guide 329 ...

Page 332: ...ontamination process Important If you have not completed the preparation steps click Cancel to stop the Decontamination wizard and perform the steps in Preparing for the Decontamination Process on page 326 before continuing 4 To view the decontamination progress details click the down arrow on the Decontamination button 330 ZE5 Cell Analyzer and Everest Software ...

Page 333: ...Decontaminating the System User Guide 331 ...

Page 334: ...h bottles containing 200 ml of DI water 6 After replacing the bottles click Continue 7 Approximately 190 min into the procedure the system prompts you to replace both sheath bottles with bottles filled to the Fill Line with sheath fluid and replace the additive and cleaner bottles with the default original bottles filled to the Fill Line with the appropriate solutions 332 ZE5 Cell Analyzer and Eve...

Page 335: ...cing the bottles click Complete Everest Software initiates a fluidics startup and then a fluidics shutdown to complete the process This can take an additional 12 min After decontamination completes the user login window appears User Guide 333 ...

Page 336: ... surface could significantly affect the light passing though When handling filters always wear gloves to avoid depositing oils and particles on the filter surface To clean an optical filter 1 Remove the filter from the instrument 2 Gently spray compressed air on the surfaces of the filter to remove any large debris particles 3 Using a lint free wipe such as Kimwipes or camera lens paper or swab mo...

Page 337: ...e baseline Re baselining establishes a new median range for CV and PMT voltages in each channel for the new bead lot Dyed beads have inherent variation so calibration must be performed for each lot The system automatically recalibrates the instrument after the bead swap is initiated Important Do not mix the contents of QC bead bottles When replacing QC beads do not add drops from the previous bott...

Page 338: ...s n If you replace the QC bead bottle with a bottle that is not full clear the Reset bead volume to full checkbox If the bottle you insert is full ensure the checkbox is selected the default setting n If you replace the QC bead bottle with a bottle from another bead lot select Change bead lot number and enter the number in the text box 336 ZE5 Cell Analyzer and Everest Software ...

Page 339: ...4 Click Continue The recalibration process verifies the new median range for CV and PMT voltages in each channel for the new bead lot against the previous QC baseline set by the administrator If the instrument recalibration with new bead lot passes no report appears The system is ready to use If the instrument recalibration with the new bead lot fails a New Bead QC report appears User Guide 337 ...

Page 340: ... become the new baseline n Keep Prior Defaults and click Apply The previous CV and PMT voltage baseline values are retained for all parameters Maintaining the System When Not in Use Flow cytometry instruments have fewer performance issues if they are run and maintained regularly with no long periods between usage Everest Software includes a vacation mode which you can use to schedule regular and a...

Page 341: ...roubleshooting problems with the system Bio Rad Technical Support might ask you to provide these files so that they can better assist you in resolving problems The Everest Help and Information menu provides a quick way to export all logs generated by the system in the last 180 days To export and view system log files 1 Click the Help and Information menu button in the upper right corner 2 Select L...

Page 342: ...st telemetry and FCS files that you want to retain See Exporting FCS and RLST Data Files on page 288 for more information To delete acquired data files 1 In the Recent Experiment Sessions panel in the Home window expand the experiment that contains the data you want to delete for example 5Color_2 2 In the expanded experiment locate and note the name of the specific session By default the session s...

Page 343: ...you recently acquired for the session 7 If you have more than one GUID folder open each folder and locate those that contain a folder labeled fcs 8 Delete the GUID folder or folders that contain an fcs folder 9 Log in to Everest Software Your experiment appears in the Recent Experiments panel You can edit and run the experiment and acquire data again Note Because you deleted the acquired data Ever...

Page 344: ...ument listed as ZE5 3 On the FlowJo toolbar click Preferences 4 In the Preferences window select Cytometers 5 In the left column select GENERIC ZE5 6 Optional In the Cytometer Identification section change GENERIC to BIORAD_ZE5 7 In the Parameter Scale Settings section clear the Custom log scaling and Custom linear scaling checkboxes if they are selected 8 Enter the following parameters n For Cust...

Page 345: ...efer to the figures in Replacing Sheath Bottles on page 146 Software Issues Error Possible causes Troubleshooting steps Unable to click the Play button Sample back flushing or previous run not complete Wait for the process to complete Run list error Validate that everything has been properly selected when setting up a run list Loader door open or sample plate inserted incorrectly 1 Stop the acquis...

Page 346: ...s testing after each to determine whether the problem has been resolved 1 Check the level of QC calibration beads and replace the bead bottle if needed See Replacing the QC Beads on page 335 2 Recalibrate the probe using custom media settings if needed See Media Selector on page 69 Data suddenly disappear from the plots in the workspace and threshold plot and the event rate drops to 0 during acqui...

Page 347: ...background fluorescence in the sample Run the cleaning process using bleach See Cleaning the Sample Line and Probe on page 324 If this does not resolve the problem contact Bio Rad Technical Support for assistance Flow cell dirty Ensure that the flow cell is clean Built up cellular material on the cuvette walls can cause unexpected light scatter which can then be introduced into the scatter detecto...

Page 348: ...nfirm Filter Choices on page 135 PMT malfunctioning Contact Bio Rad Technical Support for help Mirror or filter scratched Inspect and replace if necessary See Working with Optical Filter Configurations on page 120 Contamination of flow cell if PMT change is seen in FSC channel Follow these steps testing after each to determine whether the problem has been resolved 1 Run the cleaning process See Cl...

Page 349: ... do not fluoresce in certain channels especially for the UV and violet lasers Flow cell clogged Run the unclog process See Unclogging the Sample Line and Probe on page 322 Flow cell dirty Follow these steps testing after each to determine whether the problem has been resolved 1 Run the cleaning process See Cleaning the Sample Line and Probe on page 324 2 Run the Decontamination wizard See Decontam...

Page 350: ...tion Mode Controls on page 282 Event rate lower than expected based on sample concentration Flow cell is clogged Run the unclog process See Unclogging the Sample Line and Probe on page 322 Miscalibration of media sample probe not going into sample fully Recalibrate the probe using custom media settings if needed See Media Selector on page 69 Sample has settled Modify the run list to include an agi...

Page 351: ...r voltage and threshold value until data appear as expected in the trigger channel plot in the workspace See Configuring Instrument Settings on page 257 High voltage present in certain channels during QC process Incorrect beads used Ensure single peak QC Beads are being used ProLine Rainbow Beads or ProLine Universal Calibration Beads do not fluoresce in certain channels especially for the UV and ...

Page 352: ...nd Probe on page 324 3 Run the Decontamination wizard See Decontaminating the System on page 325 If these steps do not resolve the issue contact Bio Rad Technical Support Air in system Stop sample acquisition and run a stat tube filled with at least 500 µl DI water Dirty optical filters Inspect and clean filters See Cleaning the Optical Filters on page 334 Improper laser delay Run the QC process a...

Page 353: ...epare a new sample Cells have naturally high auto fluorescence Adjust PMT voltages to place cells on scale See Configuring Instrument Settings on page 257 Poor compensation Run the compensation process See Adjusting Compensation Automatically on page 295 Bacterial contamination causing autofluorescence Run the Decontamination wizard See Decontaminating the System on page 325 Secondary antibody cro...

Page 354: ...68 Laser or lasers not functioning properly Contact Bio Rad Technical Support for help Events below threshold or threshold set too high Adjust the trigger PMT voltage or decrease the threshold percentage See Configuring Instrument Settings on page 257 Threshold not set correctly Change threshold value See Configuring Instrument Settings on page 257 PMTs set too high or too low to see data Edit PMT...

Page 355: ...door and check the sample chamber for leaks Ensure that the probe is moving correctly Contact Bio Rad Technical Support for assistance with checking for leaks in the sample line connection Incorrect optical filter in place Run the ZE5 EYE process to ensure that all filters are correct and in the right locations See Using the ZE5 EYE to Confirm Filter Choices on page 135 Sample too dilute Recreate ...

Page 356: ...der door open Stop the acquisition process close the loader door and proceed with running the experiment No events present during QC process Probe position not sufficient to aspirate low fluid levels in QC bead bottle Contact Bio Rad Technical Support for assistance with recalibrating the probe position for the bead station Bead bottle empty Replace bead bottle See Replacing the QC Beads on page 3...

Page 357: ...2 Run the Decontamination wizard See Decontaminating the System on page 325 Debris in sheath or DI water bottles Follow these steps testing after each to determine whether the problem has been resolved 1 Run the cleaning process See Cleaning the Sample Line and Probe on page 324 2 Run the Decontamination wizard See Decontaminating the System on page 325 Dead cells in sample Adjust gates see Applyi...

Page 358: ... Evaluate sample preparation and experiment setup Inadequate cell preparation Ensure adequate cell separation and preparation because multiple cell types or debris could be present in a sample Large number of doublets in sample Adjust the flow rate down Unexpected fluorescence signal Free dye accumulating in sample line Run the cleaning process See Cleaning the Sample Line and Probe on page 324 35...

Page 359: ... and experiment setup Poor compensation Run the compensation process See Adjusting Compensation Automatically on page 295 Reagent old or degraded Antibody might not have been stored in the proper conditions refrigerated and kept in the dark Antibodies are not compatible Verify that the secondary antibody used has been grown against the species in which the primary antibody has been grown Lasers tu...

Page 360: ...ngs See PMT and Laser Controls on page 258 Probe is lowered into a tube or well when a run list starts but the run list immediately stops Probe crash Check the position of sample device Ensure that tubes are set properly in the rack and that the rack or plate is flush against the loader Recalibrate the probe using custom media settings if needed See Media Selector on page 69 Incorrect plate insert...

Page 361: ... the following fluorophores n Alexa Fluor 488 n Brilliant Ultraviolet BUV 395 n APC Cy7 n PE R phycoerythrin n PE Cy7 n PE Dazzle 594 n Brilliant Violet BV 421 n Brilliant Violet BV 510 n Brilliant Violet BV 711 Preparing Controls and Samples First prepare the compensation controls and samples to be placed in your medium of choice such as a 40 tube rack 1 Prepare a compensation control for each fl...

Page 362: ...Home window click New Experiment 2 Enter the name for the experiment and click the Next arrow 3 In the Selected Fluorophores pannel double click fluorophore names in the Fluorophores list to add them to the Selected Fluorophores list 4 In the Available Detection panel change the parameter names to reflect the cell markers used in the experiment 5 Click the Next arrow 360 ZE5 Cell Analyzer and Ever...

Page 363: ...rol to indicate that the experiment will use a negative control The first position in the compensation template is automatically assigned to the negative control as shown in the next figure 8 In the Template area of the Selected Fluorophores panel click Compensation to automatically add compensation positions for each fluorophore in the experiment User Guide 361 ...

Page 364: ...hores as shown in the next figure Setting Up the Sample and Sampling Parameters Next set up a fully stained sample and specify settings such as gate limits agitation time and target flow rate or target event rate 1 In the Plate Setup pane select an unused position and then click Sample for the position type 362 ZE5 Cell Analyzer and Everest Software ...

Page 365: ...Setting Up the Sample and Sampling Parameters 2 Optional To change the sample name click the name box in the Sample Naming pane and type a new name User Guide 363 ...

Page 366: ...e positions in the template b Select the Gate checkbox The volume defaults to the maximum volume for the media type you are using You will set sample and control gate limits in the Settings screen after completing the plate setup c In the Volume box enter the actual volume used in the tubes for this experiment 364 ZE5 Cell Analyzer and Everest Software ...

Page 367: ... rate or target event rate 4 Click the position for the last compensation control 5 Click Pause After to instruct the run list to pause after the last compensation control is acquired This allows you to perform automatic compensation before running the fully stained sample A pause image appears on the selected position for example User Guide 365 ...

Page 368: ...sample setup is complete click the Next arrow Plots are created automatically for the controls Creating Plots for the Experimental Sample 1 Click the sample position in the plate map and click Create Density Plot 2 Create a forward scatter versus side scatter plot FSC SSC for the 488 laser 366 ZE5 Cell Analyzer and Everest Software ...

Page 369: ...plots will display compensated data 4 Create a region in the FSC SSC plot This region will be used to apply a gate limit to the other plots 5 In the Gate area of the toolbar apply a gate limit to the sample by typing a value in the Limit box and then clicking Sample This gate limit will be the stop limit for sample acquisition User Guide 367 ...

Page 370: ...ed to FCS files After making adjustments you can proceed to Acquisition mode 1 Load the controls and samples onto the instrument 2 Click Apply 3 Click the first position in the template This is the position from which the instrument will start sampling 4 In the Instrument Control panel ensure that Setup mode is active 5 Click Play The system displays plots in the workspace 368 ZE5 Cell Analyzer an...

Page 371: ...opulation 8 After setting up gates and voltages click Record to save data for the sample This clears existing data disables PMT adjustment and starts saving a data file Data are acquired until the set limit is reached or until you click Stop sampling does not proceed automatically to the next position 9 Repeat Step 5 through Step 8 for other samples as needed User Guide 369 ...

Page 372: ...mit is reached 12 To adjust a gate during the run click inside the region and drag the gate to include the relevant part of the sample Because Pause After was selected after acquiring data for the controls the instrument pauses to allow you to apply auto compensation before running the fully stained sample Note A check mark appears on the control positions in the Instrument Control panel to show t...

Page 373: ... regions n Let the automatic compensation process perform region determination manually adjust the regions afterward if needed The following steps illustrate the first option 1 Click a control position to view the data from that control 2 For each control ensure that the positive and negative populations are identified properly for example two populations should appear and the scatter gate set sho...

Page 374: ...ormed in the Analysis tab and those settings are moved back to the instrument compensation settings will be saved within the FCS files for any samples that are subsequently acquired If all sample acquisition is done before compensation is performed compensation settings will not be saved within the FCS files 372 ZE5 Cell Analyzer and Everest Software ...

Page 375: ...d in accordance with FCS 3 1 standards and can be analyzed using third party software 1 Click the down arrow on the Compensation button to select auto compensation options 2 Select the Prevent Automatic Region Determination checkbox to include any regions that you adjusted manually 3 Click Calculate This applies auto compensation using the data from the compensation controls and refreshes the plot...

Page 376: ...e calculated compensation values back to the local instrument Everest software displays the contents on the Acquisition tab 5 On the Acquisition toolbar click View Compensation Matris The compensation matrix displays the calculated compensation values 374 ZE5 Cell Analyzer and Everest Software ...

Page 377: ...hat Acquisition Mode is active 2 In the plate map click the sample position from which acquisition should resume 3 Click Play to start acquiring data from the fully stained sample 4 To ensure that the gate is applied to all of the plots click the filter tool in each plot and select R1 The filter is applied to each plot User Guide 375 ...

Page 378: ...On the Analysis tab click Export analyze data using third party software Tip You can also export data from the Local Instrument tab 3 Select an export option and click Export Note Everest Software exports one FCS file per acquired control or sample For more information about exporting data files see Exporting FCS and RLST Data Files on page 288 376 ZE5 Cell Analyzer and Everest Software ...

Page 379: ...rting Final Data 4 In the Browse For Folder dialog box that appears accept the default Export folder or navigate to another location and click OK 5 Close the export dialog box when the export is complete User Guide 377 ...

Page 380: ...Appendix B Example 9 Color Immunophenotyping Experiment 378 ZE5 Cell Analyzer and Everest Software ...

Page 381: ...ater or waste line to the instrument Caution Only Bio Rad Service personnel are permitted to connect the ZE5 Cell Analyzer to external DI water or waste lines Connecting External DI Water When the instrument is connected to external DI water and is put into external DI water mode the volume in the top DI water bottle will drop to around 1 4 full while sheath fluid is flowing then the bottle will a...

Page 382: ...ter supply must be of a pressure between 10 and 20 psi filtered by a 1 μm or better filter and be of pure deionized water with at least 10 megaohm cm resistivity The next two figures show a DI water hose connected to the instrument s external DI water port and the DI water hose connected to a house DI water filter system respectively 380 ZE5 Cell Analyzer and Everest Software ...

Page 383: ...l the External Waste kit and connect the ZE5 Cell Analyzer to external DI water or waste lines The waste contains samples that are run through the instrument and thus could contain biohazardous materials Consult with your local safety officer or review local state and federal regulations to ensure proper handling and disposal of biohazardous substances Specifying External Port Use After the instru...

Page 384: ...Appendix C Optional External DI Water and Waste Ports 382 ZE5 Cell Analyzer and Everest Software ...

Page 385: ...esolution with standard FSC detector 0 3 µm FSC resolution with small particle detection module Loader Integrated sample loader with agitation Sample and collection temperature control from 4 37 C Peltier solid state system Media types n 5 ml tubes 12 x 75 mm 1 40 tubes per rack n 1 5 ml tubes 1 24 tubes per rack n 96 well plates n 96 deep well plates n 384 well plates n Stat tube position for sin...

Page 386: ...onics Speed 100 000 events per second with all parameters enabled Data processing Simultaneous measured peak area and width for every channel 24 bit data for peak and area 17 bit data for width with high resolution linear interpolation at the half height Fluidics Bulk fluids Four 4 L bulk fluid bottles onboard for sheath fluid and waste Onboard sheath additive concentrate and cleaner Optional kit ...

Page 387: ...low Cytometry Standard FCS format FCS 3 1 QC Automated quality control with onboard calibration beads Monitor 29 LCD 2560 x 1080 resolution Printer Optional Installation Power AC 96 264 V 50 60 Hz 500 W Dimensions instrument only W x D x H 29 x 27 x 26 in 74 x 66 x 69 cm Weight instrument only 260 lb 118 kg Temperature Ambient temperature range 18 25 C Relative humidity 20 60 Air and vacuum supply...

Page 388: ... Subpart B Class A Environmental EN 50581 2012 Laser IEC 60825 1 2014 EN 60825 1 2014 Class 1 laser product per IEC 60825 1 and CDRH requirements and regulations Safety IEC 61010 1 2010 EN61010 1 2010 IEC 61010 2 081 2015 EN61010 2 081 2015 UL CSA 61010 1 2012 Use For research use only Table 44 ZE5 Cell Analyzer specifications continued 386 ZE5 Cell Analyzer and Everest Software ...

Page 389: ...er 17 color 488 561 640 Computer System 17002096 Includes n ZE5 Cell Analyzer Computer with Network Adaptor n ZE5 Cell Analyzer Computer Monitor 29 in 2560 x 1080 n ZE5 Cell Analyzer Wireless Keyboard and Mouse Accessory and Replacement Parts 12004273 ZE5 Cell Analyzer Accessory Kit 12004397 ZE5 DI Water Container with Tubing blue cap 4 L 12004418 ZE5 DI Water Container 4 L pack of 2 12004396 ZE5 ...

Page 390: ...40 x 5 ml 12004446 ZE5 Tube Rack 24 x 1 5 ml 12004389 ZE5 Webcam Logitech C310 with USB cable Consumables 12004274 Test Tubes 5 ml 12 x 75 mm pack of 25 12004272 Cytometer Cleaner 1 L 12004271 ZE5 Additive 4 x 300 ml 12004403 ZE Series QC Beads 5 ml pack of 3 12005773 96 well Microplates polystyrene U bottom clear 10 pieces bag 1451083 ProFlow Sort Grade Water 5 x 4 L 1451085 ProLine Rainbow Beads...

Page 391: ...hn Wiley Sons n Cytometry Part A Journal of the International Society for the Advancement of Cytometry Wiley http onlinelibrary wiley com journal 10 1002 28ISSN 291552 4930 n Purdue University Cytometry Laboratories cytometry and confocal microscopy education and research material cytometry email archive and links to cytometry web sites and suppliers worldwide http www cyto purdue edu User Guide 3...

Page 392: ...Appendix F References 390 ZE5 Cell Analyzer and Everest Software ...

Page 393: ...f data defined within the time in seconds specified by the user Data are automatically refreshed Cycle mode is primarily used while adjusting PMT voltages regions and gates in Setup mode dichroic filter An optical component usually part of the detection path that allows passage of a range of wavelengths of light while reflecting the rest Dichroic filters can either be longpass or shortpass event A...

Page 394: ... lasers intersect the sample core inside the flow cell longpass filter An optical component usually placed in front of a detector that allows passage of a range of wavelengths of light above a specified wavelength while absorbing or reflecting the rest neutral density filter An optical filter that reduces or modifies the intensity of all wavelengths of light equally by reflecting or absorbing a po...

Page 395: ...very single particle that passes through the interrogation point samples contain debris that would inundate the dataset and drown out the population s of interest Additionally the threshold is helpful in eliminating irrelevant optical noise from the data time parameter Each event is time stamped when analyzed All events can be viewed with respect to when they occurred during the sample acquisition...

Page 396: ...feature that verifies the configuration of the optical filter setup by using multiple LEDs to pulse ten different wavelengths of light into the optical filters that lead to the detector banks 394 ZE5 Cell Analyzer and Everest Software ...

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