BD FACSDiva Option Users Guide

BD FACSDiVa Option Users Guide
http://www.bdbiosciences.com/ Part No. 334556 Rev. A November 2002
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2002, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD Biosciences. The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer input on corrections and suggestions for improvement. BD, the BD logo, BD CaliBRITE, BD CellQuest, BD FACSComp, BD FACSConvert, BD FACSDiVa, BD FACStation, and BD FACSVantage are trademarks of Becton, Dickinson and Company. PerCP is licensed under US Patent No. 4,876,190; Cy5.5 is licensed under US Patent Nos. 5,268,486; 5,486,616; 5,569,587; 5,569,766; and 5,627,027; APC and PE are licensed under US Patent Nos. 4,520,110; 4,859,582; 5,055,556; European Patent No. 76,695; Canadian Patent No. 1,179,942. Adobe and Acrobat are registered trademarks of Adobe Systems Incorporated. FlowJo is a trademark of Tree Star, Inc. Java is a trademark of Sun Microsystem, Inc. in the US and other countries. Macintosh is a trademark of Apple Computer, Inc., registered in the US and other countries. Microsoft and Windows are registered trademarks of Microsoft Corporation. ModFit LT is a trademark of Verity Software House, Inc. Sentinel SuperPro is a trademark of Rainbow Technologies, Inc. Sybase, Adaptive Server, Adaptive Server Anywhere, and SQL Anywhere are trademarks of Sybase, Inc or its subsidiaries. All other company and product names might be trademarks of the respective companies with which they are associated. This product is for Research Use Only. Not for use in diagnostic or therapeutic procedures.
History
Revision 341756 Rev A 330798 Rev A Date 8/01 1/02 Change Made Production release for software version 1.0. Updated for software version 2.0: enhanced performance, database redesign and data management utility, scalable data display, instrument settings features, Next button, more copy/paste ability, plot display features. Refer to the ReadMe file for details. Updated for software version 2.1: enhanced performance, workspace redesign with separable components, Browser-level folders, functioning Acquisition pointer, Sort Layout redesign, objects duplicated by dragging, drill-down gating, log decade gridlines on plots, view/hide gate boundaries, context-sensitive cursors, histogram smoothing, gate changes downloaded during sorting, automatic acquisition during record/sort, Experiment import/export, Ratio Scaling factor per ratio, Area Scaling factor per laser. Refer to the ReadMe file for details. Instrument features and operation separated from general software information. Instrument procedures updated to reflect version 2.2 of the software. Refer to the BD FACSDiVa Software Users Guide for more information.
330802 Rev A
05/02
334555 Rev A
11/02
Contents
About This Guide Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using Microsoft Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 1: Features of the BD FACSDiVa Option About the BD FACSDiVa Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BD FACSDiVa Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BD QuadraSort Tube Holder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BD FACSDiVa Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 2: Instrument Setup and Optimization Starting Up the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Instrument Optimization and Quality Control . . . . . . . . . . . . . . . . . . . . . . . Preparing the Alignment Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optimizing Signals from the Primary Laser . . . . . . . . . . . . . . . . . . . . . . Optimizing Signals from the Second-Laser Intercept . . . . . . . . . . . . . . . . Optimizing Signals from the Third-Laser Intercept . . . . . . . . . . . . . . . . . Reusing the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii viii ix ix 11 12 13 14 17 18 21 22 24 24 25 30 37 44 50
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Chapter 3: Running Samples Performing Sample Optimization Using Instrument Setup . . . . . . . . . . . . . . . Creating the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adjusting the Voltages and Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . Calculating Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recording and Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up the Acquisition Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recording Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reusing the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saving the Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 4: Sorting Sorting Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sort Setup Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sort Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sort Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict Resolution with BD FACSDiVa Software . . . . . . . . . . . . . . . . . . . . Yield Mask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Purity Mask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phase Mask . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sort Precision Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Sorting Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up for Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Main Sorting Adjustments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up for Sorting Into Test Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Installing the Sorting Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adjusting Sort Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51 52 54 56 59 61 61 63 64 67 67 69 70 71 77 83 84 85 86 87 88 90 90 92 92 92 95
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Calculating the Drop Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defining the Bead Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sorting Beads to Determine the Drop Delay . . . . . . . . . . . . . . . . . . . . . . Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Starting and Monitoring the Sort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up for Sorting Into a Plate or Slide . . . . . . . . . . . . . . . . . . . . . . . . . . Installing the Sorting Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adjusting the Home Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Creating a Custom Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 5: DNA Analysis Criteria for DNA Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CEN Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Running CEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CTN Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Running CTN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optimization for Data Recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 6: Calcium Flux Intracellular Calcium Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Calcium Flux Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Using the Time Parameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Optimizing the Calcium Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Measuring Calcium Flux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
100 100 100 102 104 104 106 106 107 108 110 113 114 115 116 118 122 122 125 127 128 129 129 130 133 135 136
Contents
Chapter 7: Troubleshooting Electronics Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acquisition Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sorting Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A: Optical Configurations Six-Color Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternate Six-Color Configuration: Five Colors + DNA . . . . . . . . . . . . . Seven-Color Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eight-Color Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternate Eight-Color Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . Configuration Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Index
139 140 142 149 151 152 153 154 155 156 157 159
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About This Guide

This guide describes how to operate the BD FACSVantage SE flow cytometer in digital mode using BD FACSDiVa software. For information about using the software, refer to the BD FACSDiVa Software Users Guide. BD FACSVantage SE cytometers modified with the BD FACSDiVa option can be operated in analog mode as described in the BD FACSVantage SE Users Guide. Even when using the instrument in the digital mode, consult the instrument users guide for descriptions of instrument components; daily shutdown, maintenance, and troubleshooting; theory of operation; and laser service procedures. The BD FACSDiVa Option Users Guide assumes you have a working knowledge of basic Microsoft Windows operation. If you are not familiar with the Windows operating system, refer to the documentation provided with your computer. Before using the BD FACSDiVa option, review the BD FACSDiVa ReadMe file by double-clicking the shortcut on the Windows desktop. The file contains important information that is not printed in this users guide. First-time users of the BD FACSDiVa option should read Chapter 1 to learn about option components. Instructions for routine acquisition and analysis can be found in Chapters 2 and 3. For application-specific information, review Chapter 4, Sorting; Chapter 5, DNA Analysis; and Chapter 6, Calcium Flux. For a summary of optical bench layouts for the default Instrument Configurations, see Appendix A.
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Conventions
The following tables list conventions used throughout this guide.
Table 1 Notice icons
Icon Notice Type Use Describes important features or instructions Alerts you to potential loss of data or potential damage to an application, system, or device Alerts you to potential personal injury Highlights features or hints that can save time and prevent difficulties
NOTE CAUTION WARNING
Tip
Table 2 Text and keyboard conventions

Convention Italics Use Italics are used to highlight book titles and new or unfamiliar terms on their first appearance in the text. The arrow indicates a menu choice. For example, choose File > Print means to choose Print from the File menu. When used with key names, a dash means to press two keys simultaneously. For example, Ctrl-P means to hold down the Control key while pressing the letter p.
>
Ctrl-X
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Using Microsoft Windows ! Tip

To delete objects in the workspace, be sure to press the Delete key rather than the Backspace key. You will notice that the Windows mouse has two selection buttons. Most actions are performed with the left button. Press the right mouse button while clicking an icon or object to open a contextual menu. Contextual menus contain common commands that apply to the selected item. For example, by clicking an open Experiment with your right mouse button, you can choose to copy, rename, or close the Experiment.
Technical Assistance
For technical questions or assistance in solving a problem Read the section of the users guide specific to the operation you are performing. See Chapter 7, Troubleshooting.
If additional assistance is required, contact your local BD Biosciences technical support representative or supplier. When contacting BD Biosciences, have the following information available: product name, part number, and serial number any error messages details of recent instrument performance
For instrument support from within the US, call (877) 232-8995, prompt #2-2. For support from within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor.
About This Guide
ix
1
Features of the BD FACSDiVa Option
The following topics are covered in this chapter: About the BD FACSDiVa Option on page 12 Components on page 13
11
About the BD FACSDiVa Option

The BD FACSDiVa option adds digital capability to the current analog functionality of the BD FACSVantage SE flow cytometer. BD FACSDiVa electronics continuously digitize the cytometers signals after linear amplification. Logarithmic conversion display is achieved using lookup tables. Continuous digitization eliminates dead time, improving sort yield and facilitating better sort decisions. BD FACSDiVa electronics can process up to eight fluorescence and two scatter channels with full interbeam compensation between both height and area parameters from any laser. BD FACSDiVa software can be programmed to sort a specified number of particles from multiple gates into a variety of sorting devices, including tubes, plates, and slides. The new BD QuadraSort tube holder enables sorting into four tubes simultaneously. Digital data is processed by BD FACSDiVa software on a Microsoft Windows workstation; analog data is processed by BD CellQuest Pro software on a BD FACStation Data Management system. The computers are networked via an ethernet hub to share a printer. Refer to the BD FACSDiVa Software Users Guide for information on how digital signals are measured.
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Figure 1-1 BD FACSVantage SE flow cytometer with BD FACSDiVa option installed
Components
The BD FACSDiVa option consists of the following: digital electronics digital oscilloscope BD QuadraSort sorting hardware BD FACSDiVa workstation
Chapter 1: Features of the BD FACSDiVa Option
13
BD FACSDiVa Module
The digital electronics, oscilloscope, BD FACS AccuDrop monitor, and digital control switch are housed within the BD FACSDiVa module (Figure 1-2). The electronics are adjusted by your field service engineer during installation and do not require any user maintenance.
SAVE/RECALL
MEASURE
ACQUIRE MENU
/RESET
UTILITY
CURSOR
DISPLAY
HARDCOPY
RUN/STOP
VERTICAL
POSITION POSITION
HORIZONTAL
POSITION
TRIGGER
LEVEL
CURSOR 1 CH1 MENU
MATH MENU
CURSOR 2 CH2 MENU HORIZONTAL MENU
HOLDOFF TRIGGER MENU
VOLTS/DIV
VOLTS/DIV
SEC/DIV
SET LEVEL TO 50
FORCE TRIGGER
digital oscilloscope AccuDrop monitor

Digital Off On
5V
2mV
5V
2mV
5s
5ns
TRIGGER VIEW
Digital control switch
Figure 1-2 BD FACSDiVa module
Digital Oscilloscope
The digital oscilloscope displays the digital drop-drive waveform and the digital drop charges (Figure 1-3 on page 15). Use the digital oscilloscope to verify charging of the side streams and the amplitude level. Refer to the documentation provided with the oscilloscope for oscilloscope adjustments.
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SAVE/RECALL
MEASURE
ACQUIRE MENU
AUTOSET
UTILITY
CURSOR
DISPLAY
HARDCOPY
RUN/STOP
MEASURE Source Type
VERTICAL
POSITION POSITION
HORIZONTAL
POSITION
TRIGGER
LEVEL
digital amplitude
Source Type
CURSOR 1 MATH MENU CURSOR 2 CH2 MENU HORIZONTAL MENU HOLDOFF TRIGGER MENU
CH1 Freq
CH1 MENU
drop charge
CH1 Period CH1 None -6.12V
VOLTS/DIV
VOLTS/DIV
SEC/DIV
SET LEVEL TO 50
FORCE TRIGGER
None CH1 100V CH2 10.0V M25.0 CH1
5V
2mV
5V
2mV
5s
5ns
TRIGGER VIEW
Figure 1-3 Pulses displayed on the digital oscilloscope
AccuDrop Monitor
The AccuDrop monitor shows an image of the streams illuminated by the AccuDrop excitation source, a diode laser. Use the monitor to accurately set the drop delay and set up the streams for sorting, as described in Calculating the Drop Delay on page 100. Refer to the BD FACS AccuDrop Users Guide for specific information about the AccuDrop option.
Digital Control Switch

The Digital on/off switch determines how the instrument electronics are controlled. When switched to On, the instrument is in digital mode and the electronics are controlled by BD FACSDiVa software. When switched to Off, the instrument is in analog mode and the electronics are controlled by BD FACStation software and the instrument control panel. You can run the cytometer in either digital or analog mode, but not both simultaneously, although you can observe digital- or analog-processed data when operating in the alternate mode.
Digital Operation
During operation in digital mode, analog settings for gain, threshold, compensation, and the event rate are displayed on the analog oscilloscope. Because threshold and gain are controlled differently depending on the electronic
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mode, these settings can differ from those displayed by the digital electronics. For an accurate event count, monitor the Acquisition Status frame in BD FACSDiVa software, rather than the analog oscilloscope. When operating in digital mode, the analog oscilloscope reflects adjustments made to the photomultiplier (PMT) voltages in BD FACSDiVa software. These adjustments will also change the data displayed in BD CellQuest (or BD CellQuest Pro) software. However, analog data is subject to any gains set in BD CellQuest software and thus might differ from the data displayed on the BD FACSDiVa workstation. For this reason, it is important to use signals displayed on the analog oscilloscope and in BD CellQuest software for troubleshooting purposes only. CAUTION Because digital data processing is different from analog data processing, do not save BD CellQuest files collected while operating in digital mode. NOTE In digital mode, most controls on the instrument control panel are inactive (see Figure 1-4). Equivalent controls can now be found in BD FACSDiVa software.
DROP DRIVE CONTROLS CONTRAST
BRIGHT
H HOLD
STREAM CONTROLS
center stream control
deflection plates on/off

V HOLD
drop strobe on/off

VIEWING MARK SORT CONTROLS
stream lamps on/off
plate voltage control

ON MIN DROP # 2 CHARGE MAX MIN DROP # 3 CHARGE MAX MIN PLATE VOLTAGE MAX OFF DROP DRIVE ATTENUATION
ON OFF
INDEX SORT
Figure 1-4 Instrument controls (shaded) active in digital mode
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Analog Operation
During operation in analog mode, instrument controls on the BD FACSVantage SE control panel are enabled. Any changes made within BD FACSDiVa software will not be registered on the analog oscilloscope, nor in BD CellQuest software. However, changes made in BD FACSDiVa software will be reflected on the digital oscilloscope.
BD QuadraSort Tube Holder

The BD QuadraSort tube holder, provided with the option, allows sorting into four tubes simultaneously (Figure 1-5). The angle and height of the tubes can be adjusted to optimize sample collection during sorting.
Figure 1-5 BD QuadraSort tube holder
17
BD FACSDiVa Workstation
The BD FACSDiVa workstation controls the BD FACSVantage SE flow cytometer when operated in digital mode. It consists of a Windows 2000 computer running BD FACSDiVa acquisition and analysis software. Refer to the BD FACSDiVa Software Users Guide for complete instructions on using the software. The BD FACSDiVa workstation is operated independently of the BD FACStation workstation with a separate keyboard and mouse.
Viewing BD FACSDiVa and BD CellQuest Pro Software Simultaneously

During digital operation, BD FACSDiVa software can be expanded and viewed on both monitors simultaneously. Alternatively, as shown in Figure 1-6, signals can be viewed in BD FACSDiVa software on one monitor and BD CellQuest Pro software on the other monitor by pressing the appropriate pushbutton on the bottom of the BD FACStation monitor. Your service engineer will indicate the appropriate button during installation. NOTE The monitors included with your system might be different from those shown in the figure.
Figure 1-6 Viewing BD FACSDiVa and BD CellQuest software simultaneously
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If you are accustomed to working with analog signals, note that digital data looks very different. See Figure 1-7.
BD Symphony plot
BD CellQuest Pro plot
Figure 1-7 Digital vs analog data
CAUTION Use caution when viewing digital data within BD CellQuest software. Data viewed in BD CellQuest software results from instrument setting adjustments made in BD FACSDiVa software. Digital instrument settings are not updated in BD CellQuest instrument settings files or the resulting FCS files. BD does not recommend saving BD CellQuest files during digital operation; use BD CellQuest signals for troubleshooting purposes only.
19
20
2
Instrument Setup and Optimization
This chapter describes how to start up the BD FACSVantage SE instrument for operation in digital mode, and how to use BD FACSDiVa software to optimize the instrument before acquisition. The following topics are covered in this chapter: Starting Up the Instrument on page 22 Instrument Optimization and Quality Control on page 24 Setting Up the Experiment on page 25 Optimizing Signals from the Primary Laser on page 30 Optimizing Signals from the Second-Laser Intercept on page 37 Optimizing Signals from the Third-Laser Intercept on page 44 Reusing the Experiment on page 50
21
Before beginning this chapter, you should be familiar with the following:
General instrument setup procedures; refer to the BD FACSVantage SE Users Guide for detailed information on instrument setup. BD FACSDiVa workspace components BD FACSDiVa instrument and acquisition controls BD FACSDiVa gating and statistics tools
Refer to the BD FACSDiVa Software Users Guide for information on operating this software.
Starting Up the Instrument

Follow these instructions to start up the instrument for operation in digital mode.
1 Turn on the laser water. 2 Turn on the laser(s).

In general, allow the lasers to warm up for at least 30 minutes. Refer to the BD FACSVantage SE Users Guide for specifications for each laser.
3 Open the vacuum source and air supply. 4 Verify that the sheath container is full and the waste container is empty.
WARNING The waste container contents might be biohazardous. Expose waste container contents to bleach (10% of total volume) before disposal. Dispose of waste in accordance with local regulations. Use proper precaution and wear suitable protective clothing, eyewear, and gloves.
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CAUTION Before proceeding with step 5, verify that the BD QuadraSort tube holder is not installed. If you turn on the power with the hardware installed, sorting hardware for the BD CloneCyt Plus option could catch on the BD QuadraSort tube holder. As a result, the motor could be damaged.
5 Turn on the cytometer main power switch. 6 Switch the digital control switch to ON, if necessary.
Turn the switch to ON to operate in digital mode.
7 Turn on the computer main power switch and start up the BD FACSDiVa
workstation; start up the BD FACStation computer, if needed. After logging on to Windows, launch BD FACSDiVa software by doubleclicking the shortcut on the desktop. Verify that the instrument is connected by checking the Instrument frame in the software. The message Instrument Connected appears after the cytometer connects to the workstation (this can take several minutes). If the message Instrument Disconnected remains, refer to the troubleshooting suggestions in the software manual.
8 Switch on the main pressure toggle switch. 9 Remove the sample tube from the sample injection port (SIP). 10 Turn the Fluidics Control knob to Fill for 1020 seconds to remove air
bubbles.
11 Turn the Fluidics Control knob to Run.
Chapter 2: Instrument Setup and Optimization
23
Instrument Optimization and Quality Control

Instrument optimization is a process that ensures consistent instrument performance on a daily basis given the same laser power, alignment sample, and instrument settings. Daily instrument optimization consists of the following: copying instrument settings from a previous, similar setup running an alignment sample (beads or prepared cells) optimizing signals from the laser beams
When instrument settings and the alignment sample are kept constant, changes in the means and CVs indicate variations in instrument performance over time. Keep track of means and CVs in a quality control (QC) log. QC data should be analyzed for trends over the past 3060 runs. NOTE QC results are affected by laser and fluidics performance. BD Biosciences strongly recommends following the laser and fluidics maintenance procedures in the BD FACSVantage SE Users Guide. CAUTION Do not place heavy objects or lean on the instrument console while performing this procedure. Unnecessary pressure on the instrument during or after instrument optimization could disrupt alignment, and it would have to be performed again.
Preparing the Alignment Sample

Choose an alignment sample that gives a consistent signal and is readily available, such as chicken red blood cells (CRBCs) or alignment beads. Make sure the alignment sample can be excited by your systems lasers and that the appropriate filters are installed to detect the alignment signal(s). Prepare the alignment sample according to the manufacturers instructions.
24
Setting Up the Experiment

The steps in this section show you how to set up an Experiment for instrument optimization. If you have already created a similar Experiment, you can reuse it by duplicating or importing the Experiment. Refer to the BD FACSDiVa Software Users Guide for more information.
1 Choose Instrument > Instrument Configuration and verify the current

configuration. Make sure the configuration lists the parameters to be measured and that the channels correspond to the optical bench configuration. CAUTION For accurate data results, the instrument optics setup must match the current Instrument Configuration.
2 Click the corresponding Workspace tools to display the Browser, Instrument

Status, Inspector, Worksheet, and Acquisition Controls frames, as needed.
3 (Optional) Create a folder for your Experiments.

Select the Experiment icon in the Browser; press Ctrl-N to add a new folder. Rename the folder with your name. Alternatively, you can name the folder Instrument Opt or you can create an Instrument Opt folder inside another folder. Refer to the BD FACSDiVa Software Users Guide for ideas on how to organize Experiments.
! Tip
To place an Experiment inside a folder, select the folder before creating the Experiment.
25
4 Press Ctrl-E to create a new Experiment; rename the Experiment with an

appropriate name. For example, use the current month and year, Instrument Opt, or the operators initials followed by an appropriate identifier.
5 Rename the new Specimen with todays date; rename the first Tube 488 nm.
This Tube will be used to optimize signals from the first laser. Your Experiment should look similar to that shown in the figure at the right.
6 With the 488 nm Tube selected in the Browser, click on the Instr. Settings >
Parameters tab in the Inspector and delete any unnecessary parameters.
! Tip
Save space in the database by listing only appropriate parameters. For example, do not list UV parameters.
7 Deselect the Log checkboxes for FITC and PE.
NOTE When aligning with beads, you might also need to deselect the checkbox for PerCP-Cy5.5.
26
8 In the Acquisition Controls frame, set the Events to Record to 10,000 evt
and the Events to Display to 500 evt.
! Tip
Decreasing the number of displayed events will increase the data refresh rate.
9 Name the worksheet with todays date.

Click on the worksheet to display worksheet options in the Inspector. Change the name in the Name field.
! Tip
You can later duplicate the Specimen for a subsequent optimization and keep the plots for each day on a separate worksheet.
10 Create the following plots for the 488 nm Tube:

FSC vs SSC and FSC vs FITC dot plots FSC, FITC, PE, and PerCP-Cy5.5 histograms
! Tip
Use the sticky buttons feature to easily create multiple plots. Press the Control key, select a plot tool, and click multiple times on the worksheet. Each click will create a new plot. To unstick the plot tool, select another tool or press the Esc key.
11 Resize the plots so that they fill 2/3 of the worksheet.
! Tip
Use the Resize tool to resize multiple plots simultaneously. Refer to the software manual for more information.
12 Right-click the 488 nm Tube in the Browser and choose Create Statistics View.
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13 Edit the Statistics view.

Right-click the Statistics view and choose Edit Statistics View Select the Populations tab and deselect the checkboxes for #Events and %Parent.
Set up the Statistics tab to display the mean and CV for FSC and each fluorescence channel. Set Decimal Places to 1 for the CVs and 0 for the means.
Click OK.
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14 Resize the Statistics view to fill the remaining 1/3 of the worksheet.
Your worksheet should look similar to the example shown in Figure 2-1.
Figure 2-1 Instrument Optimization worksheet
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Optimizing Signals from the Primary Laser

The following controls will be used to optimize signals from the primary laser beam.
4 1 2 3 5 6 7 8 10
1 2 3 4 5 6 7 8 9 10
X controlmoves nozzle right and left Y controlmoves nozzle away from and toward you Theta lockprevents theta control from moving Z controlmoves nozzle up and down in an arc Theta controlmoves nozzle along an arc so stream moves right and left Alpha controlmoves nozzle along an arc so stream moves away from and toward you Fluorescence channel height adjustment wheelraises and lowers fluorescence objective lens FSC obscuration bar vertical adjustmentmoves FSC obscuration bar up and down Excitation Beam Focus wheelmoves beam focus lens to adjust laser beam focal point on the sample stream Fluorescence Focus control knobmoves objective lens to adjust focal point of the fluorescence image (access through the upper side door)
30
1 Set or check the Z distance.

Turn on the drop strobe. Use the camera vertical adjustment wheel to position the viewing mark at the laser intercept (Figure 2-2). If the laser intercept is not visible, adjust the Y control. Adjust the Z control to position the nozzle at the upper reference mark.
nozzle upper reference mark viewing mark
Figure 2-2 Setting the Z distance
2 Check the trajectory of the fluid stream.

The stream should be entering the center or front third of the stream aspirator. If necessary, adjust the Alpha and Theta controls to correctly position the fluid stream.
3 Install the alignment sample onto the cytometer; turn the Fluidics Control
knob to Run.
4 Set the sample differential pressure to its standard level for this alignment
procedure.
5 Verify that the green Acquisition pointer is in front of the 488 nm Tube in
the Browser; click once on the pointer to begin acquisition. Alternatively, click the Acquire button in the Acquisition Controls frame; events appear in the plots.
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NOTE During digital operation, use the analog pulses displayed on the analog oscilloscope for troubleshooting purposes only. If necessary, adjust the threshold gains and log settings in BD CellQuest or BD CellQuest Pro software to change the analog pulse display. These will not affect digital data but can help in alignment.
6 Maximize the FSC signal.

Adjust the Excitation Beam Focus wheel and Y control to obtain the highest FSC signal intensity. If the FSC signal is out of standard range, check the position of the FSC obscuration bar.
7 Maximize the FITC signal.

Adjust the X control, the Fluorescence Focus control knob, and the fluorescence channel height adjustment wheel to obtain the highest FITC signal intensity.
! Tip
If you dont see any signal, increase the PMT voltage or turn on Log before adjusting the stream controls.
8 Close the FL1 iris incrementally as you continue optimizing the FITC signal.
Continue adjusting the controls and closing the iris until the iris is completely closed.
9 With the iris closed, adjust the Y control and Excitation Beam Focus wheel
for maximum FITC signal.
10 Compare the FITC signal intensity with the iris open and closed.
You should not lose more than half the maximum FITC signal intensity with the iris completely closed.
11 Open the FL1 iris and adjust the beam splitters for maximum fluorescence
intensity. On the appropriate plots, maximize the signal for SSC, PerCP-Cy5.5, and PE.
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12 Adjust the obscuration bars for minimum FSC and SSC noise, if necessary. 13 Verify the trajectory of the fluid stream.
The stream should remain in the center or front third of the stream aspirator. If necessary, adjust the Alpha and Theta controls to correctly position the fluid stream. After adjusting the controls, repeat steps 6 through 12.
Verifying Area Scaling for the Primary Laser

BD FACSDiVa software uses area as its default parameter. The area measurement provides a complete measurement of the voltage pulse, but it can be affected by how well the laser is focused and by the sheath pressure. To ensure that the PMT works within its linear dynamic range, it is important to adjust the height and area measurements to the same magnitude. For accurate linearity, verify the Area Scaling factor each time you optimize laser signal. Refer to the software users guide for more information about area scaling.
1 Click on the 488 nm Tube in the Browser and display the Parameters tab of
the Instrument Settings Inspector.
2 Select the height (H) checkbox for the FITC parameter. 3 Change the axis on the PerCP-Cy5.5 histogram to FITC-H. 4 Click the Laser tab in the Instrument frame. 5 Adjust Area Scaling for the first laser until the FITC-A intensity is similar to
the FITC-H intensity. See Figure 2-3 on page 34 for an example.
33
Figure 2-3 Primary laser area scaling before (left) and after (right) adjustment
6 Change the FITC-H histogram plot to PerCP-Cy5.5-A. 7 Deselect the checkbox for the FITC-H parameter in the Instrument Settings
Inspector.
34
Recording and Analyzing Primary-Laser Results

1 (Optional) Adjust the voltages to display fluorescence signal between
100,000 and 150,000.
2 Verify the FITC signal with the iris closed.

Close the iris. Increase the Events to Display to 10,000 events. Create an Interval gate on the FITC histogram peak. Click Acquire. After acquiring 10,000 events, take note of the FITC signal (ie, print the worksheet or write down the FITC mean fluorescence for the peak). Decrease the Events to Display to 500 events.
3 Open the iris; Ctrl-click the Acquisition pointer to begin recording data.
Alternatively, click the Record button in the Acquisition Controls frame.
4 After recording is complete, draw an Interval gate around each peak on the
scatter and fluorescence histograms. Adjust the gate on the FITC histogram, if needed.
! Tip
Ctrl-Click the Auto-Interval Gate tool and create all Interval gates. Then press Escape to unstick the button, and readjust the gates as needed.
5 Use the Population Hierarchy to rename each population defined by the

Interval gates. To display the Population Hierarchy view, right-click the plot and choose Show Population Hierarchy. Select a population in the Hierarchy view and enter a new name to change it. For example, change P1 to FSC p1, P2 to FSC p2, and P3 to FITC. See Figure 2-4 on page 36.
35
6 Edit the Statistics view to show only the named populations.

Right-click the Statistics view and choose Edit Statistics View. Click on the Populations tab and deselect the checkbox for the All Events population.
7 Copy the results into the QC log and print the worksheet for your records.
Keep a record of the primary laser results for future reference. See the following figure for an example.
Figure 2-4 Primary laser optimization using CRBCs
36
Optimizing Signals from the Second-Laser Intercept

If you are using more than one laser, you will need to optimize signals from the second laser, third laser, or both lasers after optimizing signals from the first laser beam. This section describes how to optimize signals for a standard laser configuration (primary intercept = 488 nm, secondary = 633 nm, third = UV). Refer to your BD FACSVantage SE Users Guide for more information about how the optical bench is configured. System configuration can vary greatly. If your system has a half-mirror in the OBS2 position (Figure 2-5), use this procedure to optimize the signal detected by detector option 1 (DO1) or detector option 2 (DO2), located to the left of the half-mirror. If your system has a triple-laser beam splitter in the OBS2 position, use this procedure to optimize the signal detected by DO1 or DO2, located to the right of the triple-laser beam splitter.
FL1 FL2 FL2 FL1
DO5 FL3 OBS2 = triple-laser beam splitter D03 OBS4 SSC DO4 DO2 OBS3 DO1 FL3
OBS3 DO1 DO2 SSC
OBS2 = half-mirror
one additional laser
two additional lasers
Figure 2-5 Detection of signals from the second-laser intercept
NOTE Your optical stage might be configured differently from the examples shown in the figure.
37
Preparing for Second Laser Optimization

1 Right click the dated Specimen and choose New Tube;
name the new Tube 633 nm. This Tube will be used to optimize signals from the second-laser intercept. Your Experiment should look similar to that shown in the figure at the right.
2 With the 633 nm Tube selected in the Browser, click on the Instr. Settings >
Parameters tab in the Inspector and select appropriate Area parameters.
! Tip
Save space in the database by listing only appropriate parameters. For example, do not list UV parameters. Change any listed parameter by clicking in the Parameter field and choosing a new parameter from the drop-down menu that appears.
3 Deselect the Log checkboxes for all fluorescent parameters. 4 Add additional vertical pages to the worksheet, as needed.
Click on the worksheet; change the number of pages in the Inspector.
5 Create appropriate plots for the 633 nm Tube.

For example, create the following plots: FSC vs APC and FSC vs APC-Cy7 dot plots APC and APC-Cy7 histograms
38
6 Right-click on any plot and choose Create Statistics View. 7 Edit the Statistics view to display the mean and CV for each fluorescence
channel. On the Population tab, deselect #Events and %Parent. On the Statistics tab, set Decimal Places to 1 for the CVs and 0 for the means.
8 In the Acquisition Controls frame, set the Events to Record to

10,000 events and the Events to Display to 500 events.
Optimizing Signals from the Second Laser

1 Block the third beam intercept (if present) by closing the laser shutter.
Refer to the laser manufacturers instructions.
2 Click to move the green Acquisition pointer in front of the 633 nm Tube in
the Browser; click Acquire, if needed. Alternatively, click the Next button in the Acquisition Controls frame. After starting acquisition, events appear in the plots.
! Tip
If you do not see any signal, increase the appropriate PMT voltage or change to Log. If you still do not see any signal, verify the Delay setting for the second laser (step 6 on page 40). NOTE During digital operation, use the analog pulses displayed on the analog oscilloscope for troubleshooting purposes only.
3 Adjust the appropriate beam splitter(s) to direct the 633 nm laser signal to
the appropriate PMT(s).
4 Adjust the two rear rotators (rear knobs) to obtain the highest signal
intensity and lowest CV on the fluorescence plots. The knobs are located on the beam steering prism assembly of the laser being optimized.
39
5 If necessary, adjust the two front translators (front knobs) to obtain the
highest signal intensity and lowest CV. Minor adjustment of the translators might be needed after performing laser alignment. The translators do not need adjusting on a daily basis.
6 Optimize the Delay setting for the second laser.

Adjust the delay to synchronize laser signals in time. The delay has been properly adjusted when the fluorescent signal intensity is at its highest.
! Tip
As a troubleshooting measure, estimate the laser delay setting from the analog oscilloscope using the Special Setup feature in BD CellQuest software. Draw an Interval gate around each peak on the fluorescence histograms. Use the Population Hierarchy to rename each population defined by the Interval gates. For example, change P1 to APC and P2 to APC-Cy7. Select the Laser tab in the Instrument frame. Adjust the second laser Delay setting in increments of 1 to obtain the highest mean channel for the fluorescent populations (Figure 2-7 on page 41). Change the Window Extension to zero to capture pulses within the narrowest time window.
Figure 2-6 Window Extension set to zero
40
Adjust the second laser Delay setting in increments of 0.1 to obtain the highest mean channel for the fluorescent populations.
Figure 2-7 Second laser delay before (left) and after (right) adjustment
Reset the Window Extension to the appropriate setting (typically 2). A larger Window Extension allows more flexibility for capturing pulses.
Verifying Area Scaling for the Second Laser

Because each laser has a different laser intercept height, Area Scaling needs to be verified for each laser after its signal has been optimized. Refer to the software users guide for more information about area scaling.
1 Select the height (H) checkbox for APC-Cy7 in the Parameters tab of the
Instrument Settings Inspector.
2 Create a histogram for APC-Cy7-H; draw an interval marker around the

fluorescent peak.
3 In the Laser tab of the Instrument frame, adjust Area Scaling for the second
laser until the APC-Cy7-A intensity is similar to the APC-Cy7-H intensity (Figure 2-8 on page 42).
41
Figure 2-8 Second laser area scaling before (left) and after (right) adjustment
4 (Optional) Delete the APC-Cy7-H histogram. 5 Deselect the checkbox for the APC-Cy7-H parameter in the Instrument
Settings Inspector.
42
Recording and Analyzing Second-Laser Results

1 (Optional) Adjust the voltages to display fluorescence signal between
100,000 and 150,000.
2 Adjust the Sample Differential knob to decrease the event rate to

approximately 200 events/second.
3 Ctrl-click the Acquisition pointer to record data for the 633 nm Tube.
4 Copy the second-laser results into your QC log.

Copy the fluorescence channel means into the QC log and print out the worksheet for your records (Figure 2-9).
Figure 2-9 Results for second laser optimization
43
Optimizing Signals from the Third-Laser Intercept

System configuration can vary greatly. If your system has a triple-laser beam splitter, use this procedure to optimize the signal detected by detector option 3 (DO3) or detector option 4 (DO4), located to the left of OBS2. See Figure 2-5 on page 37.
Preparing for Third Laser Optimization

1 Right-click the dated Specimen and choose New Tube; name the new Tube UV.
This Tube will be used to optimize signals from the thirdlaser intercept. Your Experiment should look similar to that shown in the figure at the right.
2 With the UV Tube selected in the Browser, click on the

Instr. Settings > Parameters tab in the Inspector and select appropriate Area parameters.
! Tip
Save space in the database by listing only appropriate parameters. For example, do not list fluorescence parameters for the primary laser. Change any listed parameter by clicking in the Parameter field and choosing a new parameter from the drop-down menu that appears.
3 Deselect the Log checkbox for all fluorescent parameters. 4 Add additional vertical pages to the worksheet, as needed.
Click on the worksheet; change the number of pages in the Inspector.
44
5 Create appropriate plots for the UV Tube.

For example, create the following plots: FSC-A vs UV1-A and FSC-A vs UV2-A dot plots UV1-A and UV2-A histograms
6 Right-click on any plot and choose Create Statistics View. 7 Edit the Statistics view to display the mean and CV for each UV channel.
On the Population tab, deselect #Events and %Parent. On the Statistics tab, set Decimal Places to 1 for the CVs and 0 for the means.

10,000 events and the Events to Display to 500 events.
Optimizing Signals from the Third Laser

1 Block the second beam intercept by closing the laser shutter.
Refer to the laser manufacturers instructions.
2 Click to move the green Acquisition pointer in front of the UV Tube in the
Browser; click Acquire, if needed. Alternatively, click the Next button in the Acquisition Controls frame. After starting acquisition, events appear in the plots.
! Tip
If you do not see any signal, increase the appropriate PMT voltage or change to Log. If you still do not see any signal, verify the Delay setting for the third laser (see step 6 on page 46). NOTE During digital operation, use the analog pulses displayed on the analog oscilloscope for troubleshooting purposes only.
45
3 Adjust the appropriate beam splitter(s) to direct the UV signal to the

appropriate PMT(s).
4 Adjust the two rear rotators (rear knobs) to obtain the highest signal
intensity and lowest CV on the UV plots. The knobs are located on the beam steering prism assembly of the laser being optimized.
5 If necessary, adjust the two front translators (front knobs) to obtain the
highest signal intensity and lowest CV. Minor adjustment of the translators might be needed after performing laser alignment. The translators do not need adjusting on a daily basis.
6 Optimize the third laser delay.

Adjust the laser delay to synchronize laser signals in time. The delay is properly adjusted when the UV signal intensity is at its highest.
! Tip

As a troubleshooting measure, estimate the laser delay setting from the analog oscilloscope using the Special Setup feature in BD CellQuest software. Draw an Interval gate around each peak on the UV histograms. Use the Population Hierarchy to rename each population defined by the Interval gates. For example, change P1 to UV1 and P2 to UV2. Select the Laser tab in the Instrument frame. Adjust the third laser Delay setting in increments of 1 to obtain the highest mean channel for the UV populations (Figure 2-10 on page 47). Change the Window Extension to zero to capture pulses within the narrowest time window. Adjust the third laser Delay setting in increments of 0.1 to obtain the highest mean channel for the UV populations (Figure 2-10 on page 47).
46
Figure 2-10 Third-laser delay before (left) and after (right) adjustment
Reset the Window Extension to the appropriate setting (typically 2). A larger Window Extension allows more flexibility for capturing pulses.
Verifying Area Scaling for the Third Laser

Because each laser has a different laser intercept height, Area Scaling needs to be verified for each laser after its signal has been optimized. Refer to the software users guide for more information about area scaling.
1 Select the height (H) checkbox for the UV2 parameter in the Instrument
Settings Inspector.
2 Create a histogram for UV2-H; draw an interval marker around the

fluorescent peak.
3 In the Laser tab of the Instrument frame, adjust Area Scaling for the third
laser until the UV2-A intensity is similar to the UV2-H intensity (Figure 2-11 on page 48).
47
Figure 2-11 Third-laser area scaling before (left) and after (right) adjustment
4 (Optional) Delete the UV2-H histogram. 5 Deselect the checkbox for the UV2-H parameter in the Instrument Settings
Inspector.
48
Recording and Analyzing Third-Laser Results

1 Adjust the voltages to display UV signal between 100,000 and 150,000. 2 Adjust the Sample Differential knob to decrease the event rate to
approximately 200 events/second.
3 Ctrl-click the Acquisition pointer to record data for the UV Tube.

4 Copy the third-laser results into your QC log.

Copy the fluorescence channel means into the QC log and print out the worksheet for your records (Figure 2-12).
Figure 2-12 Results for third-laser optimization
49
Reusing the Experiment

To reuse this Experiment, do the following.
1 Open the instrument optimization Experiment. 2 Create a new worksheet. 3 Right-click the Specimen and choose Duplicate without Data.
The three optimization Tubes appear under the new Specimen; empty plots and Statistics views appear on the new worksheet.
4 Rename the Specimen and the worksheet. 5 Double-click the 488 nm Tube to locate the plots. 6 Continue optimization.
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3
Running Samples
This chapter describes how to use BD FACSDiVa software to record and analyze sample data. If this is your first time using BD FACSDiVa software, BD recommends that you first practice the steps in this chapter using BD CaliBRITE beads. This exercise will familiarize you with digital data and help you establish target channel values. The following topics are covered in this chapter: Performing Sample Optimization Using Instrument Setup on page 52 Recording and Analyzing Data on page 61
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Before Beginning This Chapter

Before running samples, start up the instrument and optimize the electronics as described in your instrument manual. To perform the steps in this chapter, you should be familiar with the following: General instrument operation Refer to your instrument manual, if needed. General software components: workspace components, instrument and acquisition controls, tools for data analysis Review the corresponding sections of the BD FACSDiVa Software Users Guide, if needed.
Performing Sample Optimization Using Instrument Setup

Sample optimization consists of five main steps; each step is explained in greater detail in the sections that follow. It is important that you perform these steps in the order in which they are listed. You might need to vary certain steps for different sample types.
1 Create an Experiment, specify parameters, and add compensation Tubes. 2 Adjust the FSC and SSC voltages and the FSC threshold. 3 Gate the population of interest. 4 Adjust fluorescence detectors. 5 Calculate compensation.
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This section describes how to perform sample optimization using the Instrument Setup feature. Instrument Setup can be used to automatically calculate compensation settings. For more information about this feature, refer to the BD FACSDiVa Software Users Guide. If you are performing compensation manually, not all steps will apply. This section presents an example of sample optimization using a lysed, washed, whole-blood sample (LWB) stained with the following mouse anti-human antibodies: Unstained control Mouse IgG1 FITC/Mouse IgG1 PE/ Mouse IgG1 PerCP-Cy5.5/Mouse IgG1 APC/ Mouse IgG1 APC-Cy7 CD8 FITC/Mouse IgG1 PE/ Mouse IgG1 PerCP-Cy5.5/Mouse IgG1 APC/ Mouse IgG1 APC-Cy7 Mouse IgG1 FITC/CD8 PE/ Mouse IgG1 PerCP-Cy5.5/Mouse IgG1 APC/ Mouse IgG1 APC-Cy7 Mouse IgG1 FITC/Mouse IgG1 PE/ CD8 PerCP-Cy5.5/Mouse IgG1 APC/Mouse IgG1 APC-Cy7 Mouse IgG1 FITC/Mouse IgG1 PE/ Mouse IgG1 PerCP-Cy5.5/CD8 APC/Mouse IgG1 APC-Cy7 Mouse IgG1 FITC/Mouse IgG1 PE/ Mouse IgG1 PerCP-Cy5.5/Mouse IgG1 APC/ CD8 APC-Cy7
FITC-stained control
PE-stained control
PerCP-Cy5.5stained control
APC-stained control
APC-Cy7stained control
Chapter 3: Running Samples
53
Creating the Experiment

This section describes how to verify the instrument configuration, create a folder and Experiment, specify the parameters for the assay, and add compensation Tubes.

configuration. Make sure the configuration lists the parameters to be measured and that the channels correspond to the optical bench configuration. For this example, the Instrument Configuration should include the following parameters: FITC, PE, PerCP-Cy5.5, APC, and APC-Cy7. CAUTION For accurate data results, the instrument optics setup must match the current Instrument Configuration.
2 Click the corresponding Workspace buttons to display the Browser,

Instrument, Inspector, Worksheet, and Acquisition Controls frames, as needed.
3 (Optional) Press Ctrl-N to add a new folder to the Browser; rename the
folder with your name. Alternatively, you can name the folder Sample Optimization or you can create a Sample Optimization folder inside another folder.
4 Press Ctrl-E to create a new Experiment; rename the Experiment with an

appropriate name. For example, use 5-Color Expt, or your initials followed by an appropriate identifier.
! Tip
To place an Experiment inside a folder, select the folder before creating the Experiment.
54
5 Select the Experiment-level instrument settings in the Browser; click on the

Parameters tab in the Inspector and delete any unnecessary parameters (Figure 3-1). Click the selection button next to the parameter name(s) to select the rows; press the Delete key or click the Delete button in the Inspector.
Figure 3-1 Parameters for five-color optimization
6 Choose Instrument > Instrument Setup > Create Compensation Tubes.

A Compensation Specimen is added to the Experiment, along with a Stained Control Tube for each parameter in the Experiment. Worksheets containing the appropriate plots are added for each compensation Tube.
7 (Optional) Create label-specific Tubes, if needed.

Label-specific Tubes are not required for this example; they are needed only when your experiment contains samples stained with the same fluorophore conjugated to different antibodies (labels) that require different
55
compensation values. This is especially noticeable in tandem conjugates due to lot-to-lot variation. Refer to the BD FACSDiVa Software Users Guide for more information about this feature.
Adjusting the Voltages and Threshold

The unstained control will be used to check for nonspecific antibody binding; to adjust forward scatter, side scatter, and FSC threshold; to gate the population of interest (lymphocytes, in this case); and to adjust fluorescence settings.
1 Install the unstained control tube on the cytometer. 2 Verify that the green Acquisition pointer is in front of the Unstained
Control Tube in the Browser; click once on the pointer to begin acquisition. Alternatively, click the Acquire button in the Acquisition Controls frame.
3 Adjust the FSC and SSC voltages to appropriately display the scatter
properties of the LWB sample (Figure 3-2). Select the Unstained Control Tube in the Browser. Click the Instr. Settings tab in the Inspector, and then click the Parameters tab. Click the up and down arrows or drag the sliders to adjust the values.
Figure 3-2 Voltages adjusted
56
4 Click the Threshold tab and adjust the FSC Threshold, if needed.
Set the threshold to remove most of the debris without cutting off the lymphocyte population (Figure 3-2 on page 56).
5 Adjust the P1 gate on the Unstained Control worksheet to surround only

the lymphocyte population (Figure 3-2 on page 56). Select the gate by clicking on the boundary. Once selected, you can drag the gate to move it, or drag any of the selection handles to change the size and shape of the gate.
6 Select all fluorescence histograms on the Unstained Control worksheet. 7 In the Plot Inspector, select the Show Grid checkbox (Figure 3-3).
checkbox selected
Figure 3-3 Plot Inspector for fluorescent plots
In a four-log display, values are displayed from 26262,143. Thus, the first log decade ranges from 26262. Gridlines are used to delineate log decades on plots.
57
8 Optimize the voltages to place the negative population for each fluorescent
parameter within the first log decade (Figure 3-4). Refer to your instrument manual if you need assistance optimizing the fluorescent signal.
Figure 3-4 Unstained Control Tube after PMT adjustment
9 Click Record; when all events have been recorded, remove the unstained
control tube from the cytometer. NOTE Do not change the PMT voltages after the first compensation Tube has been recorded. In order to calculate compensation, all Tubes must be recorded with the same PMT voltage settings. If you need to adjust the PMT voltage for a subsequent compensation Tube, you will need to record all compensation Tubes again.
58
Calculating Compensation
Before you can calculate compensation, you will need to record data for each single-stained control.
1 Install the first stained control tube onto the cytometer. 2 Click the Next button; click Acquire, if needed.
Alternatively, move the Acquisition pointer to the next Tube. If the Start acquisition on pointer change User Preference is enabled (default option), acquisition starts automatically when the pointer is moved.
3 Adjust the P1 gate to encircle the lymphocytes. 4 Ctrl-click the Acquisition pointer to record data. 5 When recording is finished, install the next stained control tube onto the
cytometer.
6 Repeat steps 2 through 5 until data for all stained control tubes has been
recorded. Now that data has been recorded, you need to create regions around the fluorescence-positive populations on the histogram for each stained control Tube.
7 Double-click the first Stained Control Tube (FITC Stained Control) to

locate the corresponding plots on the worksheet.
8 Create an Interval gate around the fluorescence-positive population on the

histogram (Figure 3-5 on page 60). Fluorescence-negative populations were defined using the Unstained Control Tube in the previous section. If negative populations had not already been defined, you could define them by creating an additional gate around the negative population for each Stained Control Tube.
59
! Tip
Use the Auto-Interval tool to quickly create an Interval gate around the population. To keep the tool selected so you dont have to click the tool for each remaining Tube, hold down the Control key the first time you click the tool. The tool remains selected until you press Esc or select another tool.
Figure 3-5 Gating the positive population
9 Double-click the next Stained Control Tube in the Browser to locate the
corresponding plots on the worksheet.
10 Repeat steps 8 and 9 for the remaining compensation Tubes.

Once all regions have been set, you are ready to calculate the compensation.
11 Choose Instrument > Instrument Setup > Calculate Compensation.

If the calculation is successful, a dialog box appears where you can enter a name for the compensation Setup.
12 Enter a name for the compensation Setup; click OK.
! Tip
To keep track of compensation Setups, include the Experiment name, date, or both in the Setup name. The named Setup is automatically linked to the Experiments instrument settings.
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Recording and Analyzing Data

Once you have optimized the instrument electronics for your sample type, you are ready to record and analyze data. During analysis, recorded data is displayed in plots, and gates are used to define populations of interest. You can use Acquisition Templates to view and optimize data before it is recorded. BD FACSDiVa software analyzes the gated data and calculates statistics that you can print or export. This section describes how to use BD FACSDiVa software features to record and analyze sample data. As an example, data will be recorded and analyzed for two samples stained with the following reagents: CD4 FITC/CD16+CD56 PE/ CD3 PerCP-Cy5.5/CD19 APC/CD8 APC-Cy7. Two strategies are shown for reusing Analysis objects. If you are running the software on an acquisition workstation (ie, the workstation is connected to the cytometer), you can set up the analysis on an Acquisition Template, and reuse your analysis strategy for multiple samples in a run. Alternatively, Analysis objects can be copied to multiple Tubes at a time.
Setting Up the Acquisition Template

This section shows you how to use an Acquisition Template to preview and record data for multiple samples.
1 Create a new Specimen; rename the Specimen LWB. 2 Create two Tubes under the LWB Specimen; rename the Tubes
appropriately. For example, T/B/NK_001 and T/B/NK_002.
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3 Create an Acquisition Template; rename the template Record Data.

If the Default Acquisition Template is enabled in User Preferences (default option), the template is already present. Expand the Acquisition Templates folder to locate and rename the template. If the Default Acquisition Template preference is disabled, create a template by clicking the Acquisition Template tool in the Browser toolbar. You can create up to ten templates per Experiment.
4 Use the Experiment Layout dialog box to define labels and to specify the
number of events to record for each Tube. Parameter labels are defined in the Experiment Layout view. Labels will appear on the plot axes and in all statistics views. Choose Experiment > Experiment Layout. On the Labels tab, enter appropriate labels for the Tube. For example, enter CD4 in the FITC field; use the Tab key to move to the next field.
On the Acquisition tab, enter 10,000 events for Tubes 001 and 002. Notice that the Acq. tab in the Inspector updates automatically.
Click OK.
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5 On the Acquisition Template, create appropriate plots for previewing the

data. To switch to the Template view, double-click the template icon in the Browser or click the Template tool on the Worksheet toolbar. For example, create FSC vs SSC, FITC vs PE, PerCP-Cy5.5 vs PE, and APC vs APC-Cy7 dot plots.
! Tip
plots.
Ctrl-click the Plot tool to keep the tool selected until you create all
Recording Data
1 Install the first sample tube onto the cytometer. 2 Move the Acquisition pointer to the first Tube. 3 While data is being acquired, draw a region around the lymphocytes; set
the other plots to show data from the Lymphocyte population.
4 Ctrl-click the Acquisition pointer to record data. 5 When all events have been recorded, remove the tube from the cytometer. 6 Install the next sample tube onto the cytometer; move the pointer to the
corresponding Tube in the Browser.
7 Preview the data in the Acquisition Template; Ctrl-click the pointer to

record data.
8 Repeat steps 5 through 7 until data has been recorded for all tubes. 9 (Optional) Print the Experiment-level instrument settings.
Right-click the instrument settings icon and choose Print.
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Analyzing Data
This section describes how to set up plots, gates, and a statistics view to analyze the recorded data. By the end of this section, your analysis should look similar to that shown in Figure 3-6 on page 66.
1 Create a new Acquisition Template; rename the template T/B/NK Analysis. 2 Select the first Tube under the LWB Specimen and create the following plots
on the Analysis template: FSC vs SSC CD3 PerCP-Cy5.5 vs CD16+56 PE CD3 PerCP-Cy5.5 vs CD19 APC CD3 PerCP-Cy5.5 vs CD8 APC-Cy7 CD3 PerCP-Cy5.5 vs CD4 FITC
3 Create a Population Hierarchy and a statistics view and move them below
the plots on the worksheet.
4 Draw a gate around the lymphocytes; use the Population Hierarchy to

rename the population Lymphocytes.
5 Select all plots except the FSC vs SSC plot and specify to show only the
Lymphocyte population.
6 Select all plots and click the Title tab in the Plot Inspector; select the
checkboxes to display the Tube and Population names in the plot titles.
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7 Edit the statistics view to show only the Lymphocyte population and to
display the mean for all fluorochromes.
8 Draw a region around the CD3-positive population on the CD3

PerCP-Cy5.5 vs CD16+56 PE plot; name the population T Cells.
9 Draw a region around the CD16+56positive population on the same plot;

name the population NK Cells.
10 Draw a region around the CD19 population on the CD3 PerCP-Cy5.5 vs

CD19 APC plot; name the population B Cells.
11 Select the T-cell population in the Population Hierarchy view; draw a

region around the double-positive population on the CD3-PerCP-Cy5.5 vs CD8 APC-Cy7 plot, and name the population T Cytotoxic. Because the T-cell population is selected first, the T-cytotoxic cells become a subset of all T cells.
12 Select the T-cell population in the Population Hierarchy view; draw a

region around the double-positive population on the CD3 PerCP-Cy5.5 vs CD4 FITC plot, and name the population T Helper.
13 Print the analysis.

See Figure 3-6 on page 66 for an example.
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Figure 3-6 Lymphocyte analysis
66
Reusing the Analysis

Now that the analysis strategy has been defined, you can use it to analyze the remaining Tubes in the Experiment. Acquisition Templates allow you to apply an analysis strategy to a series of data files without saving the analysis each time. After previewing the data, you can print the analysis or save it to a Tube-specific worksheet (see the following section, Saving the Analysis). NOTE If you set up your Acquisition Template before recording data, you can specify to automatically save the analysis after recording data. This option is set in User Preferences.
1 Move the Acquisition pointer to the next Tube under the LWB Specimen. 2 View the data on the Acquisition Template; make adjustments to gates, as
needed. NOTE Adjustments will also apply to the next Tube that is viewed on the template. If you dont want to alter the template, save the analysis as described in the next section and make adjustments on the Tubes worksheet.
Saving the Analysis

Since the Analysis objects were created on an Acquisition Template, the analysis will not be saved with each Tube. If you want to save a copy of the analysis with any Tube, do the following.
1 Expand the T/B/NK Analysis Template in the Browser. 2 Right-click on the Analysis object and choose Copy.
Analysis object within template
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3 Click the Template tool on the Worksheet toolbar (

Worksheet view.
) to switch to the
4 Create a new worksheet for the destination Tube; rename the worksheet
appropriately.
5 Collapse the Tube in the Browser; right-click the Tube icon and choose
Paste. The elements on the template are copied to the new worksheet. You can view the analysis by double-clicking the Tube in the Browser.
! Tip
Apply the analysis to multiple Tubes by selecting more than one Tube before you choose Paste. Enable the Tube-specific worksheet user preference to automatically create a new worksheet for the pasted Analysis objects.
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4
Sorting
You can program BD FACSDiVa software to sort a specified number of particles from multiple populations into a variety of sorting devices, including tubes, plates, and slides. Specialized BD QuadraSort hardware, included with the option, provides the ability to sort into four tubes simultaneously. Up to four defined populations can be sorted into each tube, allowing up to 16 populations to be sorted at one time. Any subpopulation can be used for sorting, including populations defined by Quadrant gates, Interval gates, or derived gates. A single sort population can be defined by up to eight gates. The following topics are covered in this chapter: Sorting Controls on page 70 Conflict Resolution with BD FACSDiVa Software on page 84 General Sorting Overview on page 90 Setting Up for Sorting Into Test Tubes on page 92 Calculating the Drop Delay on page 100 Sorting on page 104 Setting Up for Sorting Into a Plate or Slide on page 106
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Sorting Controls
There are four sets of sorting controls provided with the BD FACSDiVa option. Controls on the BD FACSVantage SE control panelonly shaded controls are active in Digital mode (Refer to the BD FACSVantage SE Users Guide for a full description.)
H HOLD STREAM CONTROLS
center stream control
V HOLD
deflection plates on/off
drop strobe on/off

VIEWING MARK SORT CONTROLS
stream lamps on/off
plate voltage control

ON MIN DROP # 2 CHARGE MAX MIN DROP # 3 CHARGE MAX MIN PLATE VOLTAGE MAX OFF DROP DRIVE ATTENUATION
ON OFF
INDEX SORT
Drop Drive and side stream controls in the Sort Setup frame (See Sort Setup Controls on page 71.) Sorting instructions, controls, and counters in the Sort Layout window (See Sort Layout on page 77.) Sort Precision modes, Sort Layouts, and sorting device commands in the Sort menu Choose commands in the Sort menu for the following. Sort Precisionopens a dialog box where you can choose or define a Sort Precision mode for handling sorting conflicts; see Conflict Resolution with BD FACSDiVa Software on page 84. New Sort Layoutopens the default 2-Tube Sort Layout where other sorting instructions can be specified; see Sort Layout on page 77.
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Open Sort Layoutopens an existing Sort Layout. A Sort Layout must be selected in the Browser for this menu command to be enabled. Alternatively, double-click any Sort Layout to open it. Home Deviceopens a dialog box containing commands to move the tray support arm, either manually or to the home position; see Adjusting the Home Location on page 108. Custom Devicesopens a dialog box where custom devices can be defined; see Creating a Custom Device on page 110. Sort Reportdisplays a report showing the sort settings, acquisition counters, and Sort Layout information from the current sort. See Sort Report on page 83.
Sort Setup Controls

The Sort Setup frame contains controls used to set up the instrument for sorting. Display the Sort Setup frame by clicking the Sorting button in the Workspace toolbar ( ).
sort setup buttons
The Sort Setup frame contains drop-drive controls in the Breakoff tab and stream charging controls in the Streams tab. The values entered in each tab apply globally to BD FACSDiVa software and are not saved with Experiments or Tubes. The default values at startup are the last set of values used by the software.
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Sort setup values for different applications can be saved and recalled using the Sort Setup option in the Instrument menu.
Default settings are provided for high-, medium-, and low-pressure sorts; additional custom settings can be defined and saved. See Saving and Recalling Sort Setup Values on page 76.
Adjusting Settings
Within each tab, make adjustments using the software controls or your keyboard. To adjust a setting, click in the field containing the value you want to change, or press the indicated function key (eg, F2 for Amplitude). NOTE To use the function keys, the Sorting frame must be active (highlighted).
Do one of the following to change any value: Select the value in the field and enter a new value. Click the slider button next to the arrow keys to access a slider control. Click the pointer in the slider bar and drag it to a new value.
slider control
Use the mouse to click the up and down arrows or press the arrow keys on your keyboard to increase or decrease the values in small increments.
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Hold down the Control key while clicking the arrows or pressing the keys to increase the increments of the values.
Using the Sort Setup Buttons

The sort setup buttons control instrument functions used when setting the breakoff point and setting up the streams; thus the buttons are accessible from both tabs. Click the button to turn on or off the respective instrument function. Drop Driveturns the drop drive on or off. When the drop drive is on, values in the Breakoff and Streams tabs are sent to the cytometer. At startup, the drop drive defaults to off.
Drop Drive on
Drop Drive off
NOTE The drop drive should be off when an experiment does not involve sorting. Test Sortgenerates test side streams based on the Drop sequence specified in the Breakoff tab when the button is clicked. The drop drive must be on to enable the Test Sort button.
Test Sort on
Test Sort off
! Tip
Press the F12 key to switch Test Sort on and off.
Attenuationdecreases the amplitude of the drop drive oscillation when the button is clicked. At startup, attenuation defaults to off. As a general rule, turn on attenuation when sorting below 30 psi.
Attenuation on
Attenuation off
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Using the Breakoff Controls

Cells or particles flow through the BD FACSVantage SE nozzle tip in a fluid stream. During sorting, energy is applied to the stream to break it into droplets. Droplets detach from the stream at the breakoff point, typically a few millimeters downstream from the nozzle. Drop drive controls in the Breakoff tab are used to adjust the breakoff point by changing the characteristics of the drop drive. For examples of setting up the Breakoff tab for sorting, see Adjusting Sort Settings on page 95.
Breakoff values are used for the following: Frequencyadjusts the frequency of the drop drive from 1.0102.0 KHz; determines the number of drops formed per second. Amplitudeadjusts the amplitude or intensity of the drop drive from 0.080.0 V. Phaseadjusts the phase between drop generation and charging of the droplets from 0360 degrees. The selected value is sent to both the dropcharging electrode and the drop strobe. Drop delaysets the amount of time between when an event is measured and the breakoff point from 10140 drops. The drop delay value determines which drop will be deflected. Drop sequencedetermines the sequence of test pulses. Changes to the numerical field are not saved after restarting the application.
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Using the Streams Controls

Stream charging and trajectory are adjusted using the controls in the Streams tab. Enter a value to turn on the corresponding stream.
Far Left, Leftadjust the Far Left and Left streams by changing the amount of charge applied to sorted droplets, from 0100%. The Far Left stream is used for sorting onto a plate or slide and for four-way sorting; the Left stream is used for two- and four-way sorting. Right, Far Rightadjust the Far Right and Right streams by changing the amount of charge applied to sorted droplets, from 0100%. The Far Right stream is used for four-way sorting; the Right stream is used for two- and four-way sorting. 2nd, 3rd, 4th dropapply a correction factor for the drop charge as a percentage of the previous drop, from 100 to 100%. NOTE BD recommends that the amount of charge applied to the far left and far right streams not exceed 80%. When the charge is set to 100%, there is no remaining charge available for stream shaping via the 2nd, 3rd, and 4th drop correction factors.
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Saving and Recalling Sort Setup Values

Sort setup values (settings in the Breakoff and Streams tabs) are automatically saved when you quit the application. When you restart, the most recently used set of values is retained. You can save sort setup values for different sorting applications using the Sort Setup option on the Instrument menu. For example, you might want to define standard settings for different sorting pressures or sets of values for a two-way or four-way sort. See Adjusting Sort Settings on page 95 for an example. Choose Instrument > Sort Setup > Save As to save a set of sort setup values. Choose High, Medium, Low, or Custom, or enter a new name and click OK. NOTE Choosing High, Medium, Low, or Custom will overwrite the existing settings.
Choose Instrument > Sort Setup > Recall to switch between predefined value sets. Choose a named setup from the drop-down menu, and then click OK.
Choose Instrument > Sort Setup > Delete to delete a predefined value set. Choose a named setup from the drop-down menu, and then click OK.
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Sort Layout
A Sort Layout is a floating window containing all sorting instructions and controls. The Sort Layout designates which device will be used to collect sorted particles and which particles will be sorted into each sort location. Up to four sort counters can be displayed in the window to give ongoing status during a sort. Only one Sort Layout can be open at a time, but you can create several layouts for a single Tube, as long as each Sort Layout has a different name. Sort Layouts can also be added to Acquisition Templates. Sort Layouts are available for up to ten default collection devices; additional custom devices can be defined. (See Creating a Custom Device on page 110.)
Figure 4-1 Default Sort Layout devices
Examples of Sort Layouts for different devices are shown in the following figures; instructions for setting up a Sort Layout can be found in Setting Up the Experiment on page 104.
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collection device
sort location field for far-right Tube
sort counters sorting controls
sort location field for single well
Figure 4-2 Sort Layout for tubes (top) and for 48-well plate (bottom)
sort location field for spot on slide
Figure 4-3 Sort Layout for frosted slide
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Setting Up a Sort Layout

NOTE If an Acquisition Template is displayed in the Worksheet frame when you create a new Sort Layout, the Sort Layout will be added to the Acquisition Template, rather than the Tube. To ensure the Sort Layout applies only to the current Tube, switch to the Worksheet view before creating the Sort Layout.
1 Right-click on a Tube in an open Experiment and choose New Sort Layout.

Alternatively, select a Tube in the Browser and click the Sort Layout button in the Workspace toolbar ( ).
2 In the Sort Layout window, choose the type of device from the Device
menu. Default sorting devices are listed along with any defined custom devices. The Sort Layout window changes depending on the selected device: the number of rows and columns in the window matches the number of tubes, wells, or spots in the collection device.
3 Choose the Sort Precision mode from the Precision menu.

For more information, see Sort Precision Modes on page 88.
4 Enter the number of events to be sorted in the Target Events field.

Once defined, the number of events can be reused by choosing from the drop-down menu. For continuous sorting, choose Continuous from the Target Events menu.
5 Select the field(s) corresponding to the tube(s), well(s), or spot(s) where the
population will be sorted and choose a defined population from the Add menu. After you click in a sort location field, a menu appears where you can choose to add, delete, or clear all populations in the field (Figure 4-4 on page 80).
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Figure 4-4 Adding populations to be sorted
After you add a population, the population and the number of target events are written to the corresponding sort location field.
! Tip
Select a row or column header to select all fields in that row or column. After adding a population, it will be written to all selected fields at once.
6 Specify whether to save sort conflicts by selecting the Save Conflicts

checkbox. This checkbox is enabled only when using a two- or four-tube layout. When selected, all sort conflicts are sorted into a default location. For a two-tube layout, conflicts are sorted to the right; no other populations can be sorted to the right-most tube.
For a four-tube layout, conflicts for the Far Left tube are sorted to the left; conflicts for the Far Right tube are sorted to the right. No other populations can be sorted into the center-most tubes.
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Editing a Sort Layout

To change the number of events for any population, click on the Sort Location field(s) containing the population and then choose the population name from the Delete menu. Enter a new number in the Target Events field and then choose the population from the Add menu. To remove a population from a sort location field, select the field, and then choose the corresponding population from the Delete menu. To clear all populations from a field, select the field, and then choose Clear All.
Using Sorting Controls

Sorting controls appear at the bottom of the Sort Layout window. Use these controls to start, pause, resume, and stop sorting events.
Sortstarts sorting events for the current acquisition Tube. All counters reset to zero when this button is clicked. Events are sorted until the requested number of sorted events has been reached. Click the Sort button again to stop sorting before reaching the requested number of events; the counters stop at the number of sorted events. If you click Sort to restart sorting, the counters reset to zero.
Pausestops sorting, but not acquisition; sort counters freeze when the Pause button is clicked. Click the Pause button again to continue sorting and to continue incrementing the sort counters.
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Using Counters
Counters provide ongoing status during sorting; the fields cannot be edited. To display fewer counters in the Sort Layout window, click the View Counters button and choose a menu option. The corresponding counter is hidden. (Only counters with a checkmark next to the name are displayed.)
NOTE
Counters can be displayed only for two- or four-tube Sort Layouts.
Counters display the following information: Sort Ratenumber of events/second that met the sort criteria and were sorted Conflict Countnumber of events that met the sort criteria but were not sorted because of conflicts Conflict Ratenumber of conflicts/second Efficiencynumber of sorted events/(sort conflicts + sorted events) x 100
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Monitoring a Sort
During sorting, each sort location field displays the number of actual sorted events. When a target number is specified, the field displays the actual number of events along with the number of target events. A progress bar appears behind the Sort Rate counter field showing the progress of the sort.
Sort Report
Choose Sort > Sort Report to view a report of the current Sort Layout. This menu item is enabled only if a Sort Layout is open and the instrument is not sorting. The Sort Report can be printed or exported. NOTE After closing a Sort Layout, all counter information is lost. Thus, you should print a Sort Report immediately after sorting. A Sort Report contains the following: Header informationTube name, Sort Layout name, type of collection device, and the date and time of printing Sort settingsSort Setup values (information in the Breakoff and Streams tabs), precision mode, and masks definition Acquisition countersThreshold count, processed events count, electronic conflicts count, and elapsed time
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Sort counterscounter values per sort destination, or total sort count if sorting sequentially Sort LayoutPopulation(s), sort count, and target event count for each sort location field
The Sort Report window contains a File menu where you can choose to print or export the report. Exported comma-separated values (CSV) files can be opened with a spreadsheet application such as Microsoft Excel.
Conflict Resolution with BD FACSDiVa Software

During sorting, the cytometer deflects drops based on the characteristics of the particles in each drop and where the user wants to deflect them. Drops are deflected depending on the type of target particle, where the particle is contained in the drop, or whether the drop is free of contaminating particles. The BD FACSDiVa option accurately measures particle position to within 1/32 of a drop. Mask settings determine how drops are deflected when sorting conflicts occur. There are three Mask settings, each of which addresses a different type of conflict. These settings are combined to define sort Precision Modes: each mode is made up of a set of masks. Precision Modes are defined in the Sort Precision dialog box, accessed from the Sort menu.
84
Yield Mask
The Yield Mask setting defines how close to the edge of the drop, in 1/32-drop increments, a particle of interest can be located before sorting an additional drop. Half of each Yield Mask setting defines an equal area at each end of the drop. For example, when the Yield Mask is set to 16 and an event is within 8/32 from the beginning of a drop, the previous (leading) drop will be sorted. If an event is within 8/32 from the end of a drop, the following (trailing) drop will be sorted. See Figure 4-5.
trailing drop: sorted drop being interrogated leading drop: not sorted
Yield Mask
Yield Mask target particle
Figure 4-5 Target particle within a Yield Mask of 16
If the Yield Mask were set to 8 for the same target particle, the target particle would fall outside of the Yield Mask; thus no additional drops would be sorted. See Figure 4-6.
trailing drop: not sorted drop being interrogated leading drop: not sorted
Yield Mask
Yield Mask target particle
Figure 4-6 Target particle outside a Yield Mask of 8
When the Yield Mask is set to zero, only one drop (the drop containing the target particle) will be deflected; when the mask is set to 32, two drops will always be deflected. Yield Masks between 032 will sort either one or two drops. When more than one drop is deflected in the same direction, residual charge from the first drop will degrade the quality of the side stream. Thus, when four-way sorting or sorting into small wells where precise deflection is required, a Yield Mask of zero is recommended.
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NOTE Yield Masks cannot be used in conjunction with Phase Masks. Thus, when the Yield Mask is greater than zero, the Phase Mask automatically reverts to zero.
Purity Mask
The Purity Mask setting defines how close, in 1/32-drop increments, a contaminating drop can be located before ignoring the drop being interrogated. For example, when the Purity Mask is set to 16, the drop being interrogated will not be sorted if a non-target particle falls within the first or last 8/32 of the leading or trailing drop. In the following example, a non-target particle falls within the first 8/32, so the interrogated drop will not be sorted (Figure 4-7).
trailing drop drop being interrogated leading drop
Purity Mask
(not sorted)
Purity Mask
non-target particle
Figure 4-7 Non-target particle within a Purity Mask of 16
If the Purity Mask were set to 8 for the same target particle, the non-target particle would fall outside of the Purity Mask, so the interrogated drop would be sorted. See Figure 4-8. NOTE With any Purity mask greater than zero, the drop being interrogated must be free of contaminating particles or the drop will not be sorted.
trailing drop drop being interrogated leading drop
Purity Mask
(sorted)
Purity Mask
non-target particle
Figure 4-8 Non-target particle outside a Purity Mask of 8
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Phase Mask
Particles near the drop edge can affect the breakoff and alter the trajectory of the deflected drop. The Phase Mask restricts drop deflection when an event is too close to the edge of a drop or when there are events close to the edge of adjacent drops. A Phase Mask is used to improve counting accuracy and side-stream quality at the expense of yield. For example, when the Phase Mask is set to 16, the drop being interrogated will be sorted only if the target particle falls outside the Phase Mask (Figure 4-9).
trailing drop (drop sorted) leading drop
Phase Mask trailing drop (drop not sorted) leading drop
Phase Mask
Figure 4-9 Sorted and unsorted drop with Phase Mask of 16
Decreasing the Phase Mask to 8 allows more drops to be sorted. However, because the target particle is closer to the edge of the drop, there is more variability in drop trajectory (Figure 4-10).
trailing drop (drop sorted) leading drop
Phase Mask
Figure 4-10 Sorted drop with Phase Mask of 8
! Tip
BD recommends using a Phase Mask of at least 8 when sorting single cells.
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NOTE Phase Masks cannot be used in conjunction with Yield Masks. Thus, when the Phase Mask is greater than zero, the Yield Mask automatically reverts to zero.
Sort Precision Modes

Mask values can be combined in many different ways. By default, five Sort Precision modes are already definedPurity, Yield, Single Cell, Initial, and Fine Tune.
Precision Mode: Yield Mask: Purity Mask: Phase Mask: Single Cell:
Purity 32 32 0
Yield 32 0 0
Single Cell 0 32 16
Initial 32 0 0
Fine Tune 0 0 0
"
"
"
"
In Purity mode, the Yield Mask is set to the maximum to obtain the greatest number of particles; because the Purity Mask is also at the maximum, only drops with a target particle will be sorted. Sorting in Purity mode results in a sorted sample that is highly pure, at the expense of recovery and yield. In Yield mode, only the Yield Mask is used at its maximum value; thus recovery and yield are optimized at the expense of purity.
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In Single Cell mode, the Purity Mask is set to the maximum, so only drops containing a target particle will be sorted. The Phase mask is set at half the maximum, so only particles centered within the sorted drop are deflected. Drop trajectory and count accuracy are optimized at the expense of yield. This mode is recommended for single-cell sorting or situations where precise counting is required. NOTE Select the Single Cell checkbox to obtain the highest quality side streams and the most accurate counts. When the checkbox is selected, drops containing two target events (acceptable with a Purity Mask) are discarded. The Yield Mask is disabled when Single Cell is selected.
In Initial mode, only the Yield Mask is used at its maximum value; thus recovery and yield are optimized at the expense of purity. NOTE Initial mode is equivalent to the Yield mode. It is named differently as a reminder to use this as the initial mode when setting the drop delay using the AccuDrop option.
In Fine Tune mode, all Masks are set to zero for deflecting the maximum number of drops. This mode is used to fine-tune the drop-delay value using the AccuDrop option.
Defining New Precision Modes

Default Precision modes cannot be edited; however, you can create new modes and then choose them from the Precision Mode drop-down menu.
1 Choose Sort > Sort Precision; click Add.
The current sort mode is duplicated and the Mask fields are enabled.
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2 (Optional) Change the name of the mode in the Precision Mode field. 3 Enter values for the Yield, Purity, and Phase Masks. 4 Click to select the Single Cell checkbox, if needed. 5 Click Close.
The new mode is added to the Precision Mode drop-down menu. NOTE To delete a mode, choose it from the drop-down menu and then click Delete.
General Sorting Overview

The following section presents a general overview of the main sorting adjustments. For specific instructions, see Setting Up for Sorting Into Test Tubes on page 92 or Setting Up for Sorting Into a Plate or Slide on page 106.
Setting Up for Sorting

Follow the steps in this section to set up for sorting. Each step is explained in more detail in the context of each sorting example.
1 Install all sorting hardware, including the appropriate nozzle tip for the size
of the cells to be sorted. For specific sorting hardware, see Installing the Two-Way Sorting Hardware on page 93, Installing the BD QuadraSort Tube Holder on page 93, or Installing the Sorting Hardware on page 107. WARNING Sorting hardware could be contaminated with biohazardous material. Follow universal precautions when handling instrument hardware. As a general guideline, the nozzle tip should be six to ten times the particle diameter. Perform daily instrument optimization and quality control each time the nozzle tip is changed.
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NOTE Because the drop-delay value cannot be <10, the BD FACSDiVa option cannot sort with nozzle tips >100 m. Use one of the following nozzle tips with the BD FACSDiVa option.
Nozzle Tip Size (m) 50 60 70 80 90 100a BD Catalog No. 343592 343588 343593 343589 343591 343594
a. Not recommended for high-pressure sorting (>45 psi)
2 Start up the instrument and perform instrument optimization with

appropriate sorting hardware installed. See Instrument Optimization and Quality Control on page 24.
3 Perform sample optimization for the sample to be sorted.

See Performing Sample Optimization Using Instrument Setup on page 52.
! Tip
When sorting, perform sample optimization with the drop drive on and the frequency at an appropriate level.
4 Use gating tools and subsetting methods to define the population(s) of

interest. Examples of gating analysis can be found in Recording and Analyzing Data on page 61 and in the BD FACSDiVa Software Users Guide.
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Main Sorting Adjustments

Each of the following adjustments is explained in more detail in the context of each sorting example.
1 Adjust the (drop drive) Frequency for the shortest droplet breakoff
distance.
2 Adjust the Amplitude to optimize the last connected drop. 3 Adjust the Phase to obtain single side streams. 4 Use the AccuDrop option to determine the drop delay.
Setting Up for Sorting Into Test Tubes

This section describes how to set up for sorting into test tubes. Use this procedure as a guide to set up similar sorting experiments. For general guidelines, see General Sorting Overview on page 90.
Installing the Sorting Hardware

WARNING Sorting hardware could be contaminated with biohazardous material. Use universal precautions when handling instrument hardware.
1 Install an appropriately sized nozzle tip.

For guidelines, see Setting Up for Sorting on page 90. NOTE Instrument optimization must be performed each time you change the nozzle tip. The 100-m nozzle tip is not recommended for pressures >45 psi.
2 Install the tube holder.

For two-way sorting, see the following section. For four-way sorting, see Installing the BD QuadraSort Tube Holder on page 93.
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Installing the Two-Way Sorting Hardware

1 Verify that the Fluidics Control knob is set to Off and that the deflection
plates are not charged. WARNING To prevent shock, do not touch the deflection plates when the red warning light appears on the control panel.
2 Install the adjustable support bracket below the center-stream aspirator

using the two metal thumbscrews.
3 Install the pegs at the appropriate positions in the support bracket; slide the
collection tube holder onto the pegs. If you are using the 12 x 75-mm tube holder, ports for cooling water face toward the front of the instrument. If you are using the 15-mL tube holder, ports for cooling water face toward the rear of the instrument.
Installing the BD QuadraSort Tube Holder

The BD QuadraSort tube holder, provided with the BD FACSDiVa option, allows sorting into four tubes simultaneously (Figure 4-11). The angle and height of the tubes can be adjusted to optimize sample collection during sorting. The tube holder can accommodate any combination of eppendorf, 12 x 75-mm, and 15-mL tubes.
Figure 4-11 BD QuadraSort tube holder for 12x75-mm tubes
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NOTE When used with 15-mL tubes, the BD QuadraSort tube holder is designed to hold only polypropylene tubes (BD Catalog No. 352096), not polystyrene tubes. BD recommends placing the 15-mL tubes into the two center tube holders only, and using the outer tube holders for smaller tubes.
1 Verify that the Fluidics Control knob is set to Off and that the deflection
plates are not charged. WARNING To prevent shock, do not touch the deflection plates when the red warning light appears on the control panel.
2 Install the adjustable support bracket below the center-stream aspirator

using the two metal thumbscrews.
3 Install the pegs into the fifth and eighth holes from the top of the support
bracket; slide the BD QuadraSort tube holder onto the pegs (Figure 4-12). NOTE Optimize the position of the tube holder and the angle of the tubes for your system.
EXCITATION BEAM FOCUS
support bracket thumbscrew
peg tube holder installed
Figure 4-12 Installing the BD QuadraSort tube holder
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Adjusting Sort Settings

1 Input or recall approximate Breakoff settings for the sheath pressure.
Click the Breakoff tab in the Sort Setup frame and input approximate settings (Figure 4-13). If similar settings were saved, recall them from the Instrument > Sort Setup menu.
standard pressure
high pressure
Figure 4-13 Preliminary Breakoff settings for standard (left) and high (right) pressure
2 Click the buttons to turn on the Drop Drive and Test Sort; for standard
pressure (1012 psi), turn on Attenuation as well.
standard pressure only
WARNING To prevent shock, do not touch the nozzle when the drop drive is on or drops are being charged. In digital mode, the drop drive is on when the Drop Drive button shows a drop pattern in the Sort Setup frame; drops are being charged when the Test Sort button shows a drop pattern or when the Sort button has been clicked.
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3 Adjust the Frequency to obtain the optimal breakoff distance.

When sorting with the 70-m nozzle tip at 1012 psi, the Frequency should be in the range of 2230 kHz. For other nozzle sizes and sorting pressures, see the following table.
Nozzle Size (m) 50 70 100 Sheath Pressure (psi) 1115 1012 89 Frequency (kHz) 3234 2230 1520
When sorting with the 70-m nozzle tip at 35 psi, the Frequency should be in the range of 5577 kHz. For other sorting pressures, see the following table.
Sheath Pressure (psi) 5060 3050 2030 Frequency (kHz) 6599 5577 4055
Optimize these values for your sorting application. In general, a smaller nozzle opening requires a higher Frequency at a given sheath pressure. CAUTION The standard nozzle holder is not intended to be used at sheath pressures >20 psi. For higher sorting pressures, verify that the high-speed sort head (provided with the BD TurboSort Plus option) is installed.
4 Adjust the Amplitude to optimize the breakoff point (minimize stream noise).
! Tip
Breakoff values can be adjusted using the keyboard. Press the indicated Function key (ie, press F2 for Amplitude), and use the up and down arrow keys to adjust the value. Hold the Control key down while pressing the arrow keys to adjust the values in larger increments.
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5 Input approximate Streams settings based upon the sort direction.

Click the Streams tab in the Sort Setup frame to input settings (Figure 4-14). If you recalled settings for the Breakoff tab, verify that the Streams tab is set appropriately.
two-way sort
four-way sort
Figure 4-14 Preliminary Streams settings for a two-way (left) and four-way (right) sort
6 Adjust the phase using the digital oscilloscope.

Turn on Test Sort by clicking the Test Sort button. Press the AUTOSET button on the oscilloscope. Adjust the SEC/DIV knob until you can clearly see the digital amplitude and drop charge waveforms. Adjust the Phase value in the Breakoff tab until the drop charge is synchronized with the top or bottom of the amplitude waveform.
SAVE/RECALL MEASURE ACQUIRE MENU UTILITY CURSOR DISPLAY HARDCOPY RUN/STOP AUTOSET
digital amplitude
MEASURE Source Type

POSITION
VERTICAL
POSITION
HORIZONTAL
POSITION
TRIGGER
LEVEL
AUTOSET button
Source Type
CURSOR 1 MATH MENU CURSOR 2 CH2 MENU HORIZONTAL MENU HOLDOFF TRIGGER MENU
CH1 Freq
CH1 MENU
drop charge
CH1 Period CH1 None -6.12V
VOLTS/DIV
VOLTS/DIV
SEC/DIV
SET LEVEL TO 50
FORCE TRIGGER
None CH1 100V CH2 10.0V M25.0 CH1
5V
2mV
5V
2mV
5s
5ns
TRIGGER VIEW
SEC/DIV knob
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7 Turn the Plate Voltage knob on the instrument control panel counterclockwise to its minimum setting.
8 Turn on the deflection plates, using the push button on the instrument
control panel. WARNING To prevent shock, do not touch the deflection plates when the red warning light appears on the control panel. The plates remain energized even when the camera door is open.
9 Slowly turn up the plate voltage until the side streams are visible and
deflecting away from the waste aspirator. WARNING Do not allow the streams to touch the deflection plates because this could result in arcing (sparking). You might need to adjust the Frequency, Amplitude, Phase, or Streams settings to optimize the angle of the streams. Once optimized, the plate voltage can be increased.
10 Click the Streams tab and adjust the 2nd, 3rd, and 4th drop settings to
tighten the center stream and fine-tune the side streams.
! Tip
Generally, settings of 20, 10, and 5 are good starting values for the 2nd, 3rd, and 4th drops, respectively. If the 2nd drop must be set to zero to obtain a narrow center stream, the Frequency setting probably needs adjustment.
11 Adjust the Plate Voltage knob and Stream deflection percentages to direct
the streams into the tubes.
12 Press F12 to turn off Test Sort. 13 Calculate the drop delay.
See Calculating the Drop Delay on page 100.
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14 (Optional) Save the values in the Breakoff and Streams tabs.

Choose Instrument > Sort Setup > Save and enter an appropriate name (such as 11 psi 2-way sort) in the dialog box that appears. Click OK to save the settings. The settings can be recalled for use in a similar sorting experiment. For more information, see Saving and Recalling Sort Setup Values on page 76.
15 Print a Sort Report.

Choose Sort > Sort Report. In the Sort Report Window, choose File > Print Report.
NOTE After closing a Sort Layout, all counter information is lost. Thus, you should print a Sort Report immediately after sorting.
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Calculating the Drop Delay

Use the AccuDrop option to determine the optimal drop delay setting for your sorting application. For more information, refer to the BD FACS AccuDrop Users Guide.

The steps in this section show you how to set up an Experiment for AccuDrop optimization. Because no data is recorded, the Experiment can be reused as often as you like.
1 Create a new Experiment and rename it AccuDrop. 2 Rename the first Tube AccuDrop Beads. 3 With the AccuDrop Beads Tube selected in the Browser, click on the Instr.
Settings > Parameters tab in the Inspector and delete all parameters except FSC and SSC.
4 Create an FSC histogram for the AccuDrop Beads Tube.
Defining the Bead Population

Perform this procedure after adjusting the sort settings.
1 Move the emission filter away from the camera. 2 In the Sort Setup frame, click the button to turn on Test Sort ( 3 While viewing the streams on the AccuDrop monitor, adjust the
micrometer dial to obtain even illumination of the center and side streams. When illuminated evenly, the streams appear to sparkle (Figure 4-15 on page 101). ).
100
Figure 4-15 Illuminating the center and side streams
4 Turn off Test Sort. 5 Turn off the stream lamps to better view the streams. 6 Install a sample tube filled with a dilute suspension of AccuDrop beads
(1 drop of beads in 0.5 mL sheath fluid); click the Acquisition pointer to start acquisition.
7 Click the Parameters tab in the Inspector and adjust the FSC voltage to
place the bead population at channel 125,000.
8 Draw an Interval gate that encloses the entire histogram (Figure 4-16).
Set the endpoints of the interval at 0 and 262 x 103. CAUTION For an accurate setting, ensure that the histogram region encompasses the entire bead population, including doublets.
Figure 4-16 AccuDrop bead population
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Sorting Beads to Determine the Drop Delay

1 Right-click the AccuDrop Beads Tube, choose New Sort Layout, and set up
the Sort Layout as follows.
2 Adjust the micrometer dial to obtain the brightest bead spot on the center
stream.
3 Move the emission filter in front of the camera. 4 Adjust the Sample Differential knob to achieve a bead event rate of
approximately 4,000 events/second.
5 Click Sort in the Sort Layout window. 6 Optimize the drop delay.
In the Breakoff tab in the Sort Setup frame, adjust the Drop Delay setting until most of the beads are in the left stream and the least are in the center stream (Figure 4-17), using a 1-drop increment. Before you adjust the drop delay, the beads will appear as a bright spot on the center stream and a faint spot on the left side stream. Adjust the drop delay until the spot on the left stream is as bright as possible. This will yield the most accurate drop delay.
left stream left stream
center stream
center stream
Figure 4-17 Viewing beads on AccuDrop monitorbefore (left) and after (right) adjustment
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7 Click Sort to stop sorting. 8 In the Sort Layout, change the Precision mode to Fine Tune.
9 Repeat steps 5 through 7, adjusting the drop delay in a 0.1-drop increment.

The final drop delay setting is the optimal setting for the sort.
10 Move the emission filter away from the camera; remove the tube from the
cytometer.
11 Proceed with sorting.

See the following section.
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Sorting
Before beginning the sort, do the following: Perform sample optimization with the Drop Drive on and the Frequency at an appropriate level. Use gating tools and subsetting methods to define the population(s) of interest. Examples of gating analysis can be found in Recording and Analyzing Data on page 61 and in the BD FACSDiVa Software Users Guide.

1 If sorting into four tubes, create a 4-Way Purity Sort Precision mode.
Choose Sort > Sort Precision. Click Add. Enter 4-Way Purity in the Precision Mode field. Set the Purity Mask to 16 and the remaining masks to zero; click Close.
2 In the Browser, right-click the Tube containing the defined population

subset(s) to be sorted and choose New Sort Layout. Alternatively, select a Tube in the Browser and click the Sort Layout button in the Workspace toolbar ( ). By default, the 2-Tube Sort Layout appears.
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3 Make appropriate entries in the Sort Layout.
Choose the collection device from the Device menu. Choose the Precision mode from the Precision menu. Enter the number of Target Events by choosing a value from the dropdown menu or entering a number in the field. Select the sort location field(s) to be sorted into. Select multiple fields by dragging the mouse; select a row or column by clicking the row or column header. Add the required population(s) to each sort location field. If the Add menu doesnt appear after selecting the sort location field(s), right-click the selected fields to see the menu. Enter the number of Target Events and the population(s) for the remaining sort location fields, if necessary.
NOTE To change the number of target events in a sort location field, change the value in the Target Events field before adding the population. Once a population has been added, you cannot change the number of sorted events except by deleting, and then adding the sort population. See Editing a Sort Layout on page 81.
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Starting and Monitoring the Sort

1 Open the camera door and install the collection tubes, plate, or slide
containing nutrient medium.
2 Install the sample tube on the cytometer and close the camera door. 3 Turn the Fluidics Control knob to Run. 4 Verify that the green Acquisition pointer is indicating the appropriate Tube
in the Browser; click Sort.
5 (Optional) Click Record to save data for the Tube.

Sorting continues until the required number of cells has been sorted. If the number of Target Events is set to Continuous, sorting continues until you manually stop sorting. Monitor the sort progress from the Sort Layout window. The number of events sorted into each sort location appears in the corresponding field. The Sort Rate, Conflict Rate, and number of conflicts are displayed in the counter fields. (See Using Counters on page 82.) NOTE To pause during sorting, click the Pause button. Sort counts are retained when you restart sorting by clicking the Pause button again.
6 After completing a four-way sort, remove the BD QuadraSort sorting

hardware. CAUTION To avoid instrument damage, do not start up the instrument with the BD QuadraSort sorting hardware installed.
Setting Up for Sorting Into a Plate or Slide

This section describes how to set up for sorting into a plate or slide. Use this procedure as a guide to set up similar sorting experiments. For general guidelines, see General Sorting Overview on page 90.
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Installing the Sorting Hardware

NOTE For more details on hardware installation, refer to the BD CloneCyt Plus Users Guide.
1 Turn off the deflection plates.

WARNING To prevent shock, do not touch the deflection plates when the red warning light appears on the control panel.
2 Remove the center stream aspirator and replace it with the right and center
stream aspirator supplied with the BD CloneCyt Plus option.
3 Install the metal ground shield and the plastic tray shield. 4 Install the tray support on the support arm using the metal thumbscrew.
NOTE Install only the shorter tray support provided with the BD FACSDiVa option. Previous versions of the tray support do not allow sufficient clearance between the plate and the sort chamber.
5 Place a 96-well plate on the tray support (Figure 4-18).
EXCITATION BEAM FOCUS
right- and centerstream aspirator
plastic tray shield metal ground shield
tray support
Figure 4-18 Sorting hardware for a 96-well plate
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Adjusting the Home Location

When sorting into a 96-well plate or onto a slide, the robotic arm holding the tray support is pre-programmed to move a set interval between wells on a plate or spots on a slide. The Home location is used as the starting point: at the Home location, the far left stream should hit the center of the well in the top-left corner of the sorting device. Default Home location coordinates exist for each pre-programmed collection device. Before beginning a sort, use the following procedure to verify the Home location and adjust it, if needed.
1 Perform sort setup.

Optimize the sort settings as if you were setting up for a two-way sort at standard pressure (see Adjusting Sort Settings on page 95). All steps are identical except for configuring the Streams tab; when sorting into a plate or slide, only the far left stream is used. For step 5 on page 97, input the settings as shown in the following figure:
2 Install the collection device on the tray support. 3 Choose Sort > Home Device.
The Device Setup dialog box appears (Figure 4-19 on page 109). If a Sort Layout is currently open, the corresponding collection device will be selected in the list of devices. Otherwise, select the appropriate collection device in the list.
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Test Sort button
Figure 4-19 Setting the Home location
4 Double-click the Test Sort button to deposit a drop on the Home location. 5 Carefully remove the collection device from the tray support and note
where the drop was deposited.
6 Wipe the collection device dry, and place it back on the tray support. 7 Adjust the Home location, if necessary.
Click the appropriate Arrow buttons to move the tray support as needed. Large arrows move the tray by 5 steps; small arrows move the tray by 1 step.
8 Repeat steps 4 through 7 until the drop is centered appropriately. 9 Click Set Home, and then Close. 10 Proceed with Sorting on page 104.
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Creating a Custom Device

You can program the robotic arm to sort into any grid configuration. Create a custom device by entering the number of rows and columns and setting the Home and Farthest locations. BD FACSDiVa software calculates the increment between rows and columns to determine the sort locations.
1 Choose Sort > Custom Devices. 2 Click the Add button in the Custom Devices dialog box.
A new device is added to the list of custom devices. By default, devices are named Custom Device_00x, where x is the next consecutively numbered device (Figure 4-20).
Figure 4-20 Defining a custom device
3 Select the text in the Name field and enter a new name. 4 Enter the number of sort location Rows and Columns.
A device can have up to 60 rows and 25 columns.
110
5 Use the Arrow keys to move to the Home location; click Set Home.
See Adjusting the Home Location on page 108. There are no default values for custom devices, so more initial adjustment with the Arrow keys is required.
6 Use the same procedure to move to the Farthest location; click Set Farthest.
The Farthest sort location is the well or spot on the lower-right corner of the collection device.
7 Click Apply, then Close.

After you set the Home and Farthest locations, custom devices are listed in the Device drop-down menu in the Sort Layout window. NOTE Once custom devices are defined, you cannot change the numbers of rows and columns.
8 Proceed with Sorting on page 104.
Deleting a Custom Device

1 Choose Sort > Custom Devices. 2 Select the name of the device to be deleted from the list of custom devices
(Figure 4-20 on page 110).
3 Click Delete.
The device is deleted from the custom device list, but is retained within any Sort Layouts where it was used.
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112
5
DNA Analysis
The following topics are covered in this chapter: Criteria for DNA Experiments on page 114 CEN Optimization on page 115 CTN Resolution on page 122 Optimization for Data Recording on page 125
113
Criteria for DNA Experiments

In DNA experiments, the flow cytometer must provide the following: resolution linearity ability to distinguish singlets from aggregates
Obtaining good resolution for the DNA signal depends on proper sample preparation and instrument optimization of the optics and fluidics. The resolution of a flow cytometer can be assessed by measuring the CV of a reference particle: the lower the CV, the better the resolution. Linearity is critical for DNA experiments. To verify the linearity of DNA data, the pulse-area signal is used to measure the amount of DNA fluorescence detected from cells and nuclei. For example, the G2+M peak should be located at twice the mean channel of the G0/G1 peak (Figure 5-1).
Figure 5-1 Area signal measures amount of DNA fluorescence
Doublet discrimination, or the ability to resolve singlets from aggregates, is also important for DNA experiments. Aggregated cells or nuclei are detected as events that have two or more times the amount of singlet fluorescence. For cell-cycle analysis, it is important to resolve singlets from aggregates because doublets of G0/G1 cells have the same amount of DNA fluorescence as singlet G2+M cells. Therefore, these doublets accumulate in the same fluorescence area channel as singlet G2+M cells (Figure 5-2 on page 115).
114
Figure 5-2 Doublet discrimination
NOTE
Before beginning this chapter, do the following. Prepare biological standards for instrument quality control using the BD DNA QC Particles kit (Catalog No. 349523). Prepare one tube each of chicken erythrocyte nuclei (CEN) and calf thymocyte nuclei (CTN) sample according to the kit instructions. The CEN sample is used for instrument optimization and to check instrument resolution (CV) and linearity. The CTN sample is used to verify the systems ability to resolve singlets from aggregates.
Optimize the instrument electronics as described in Chapter 2.
CEN Optimization
Use the following procedure to set up BD FACSDiVa software for a DNA experiment that uses propidium iodide (PI) as the DNA-staining dye (eg, the BD DNA QC Particles kit). If you are using another sample type, modify the steps accordingly.
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115

configuration. Make sure that the current configuration lists the PI parameter and that the channels correspond to the optical bench configuration. The PI parameter should be assigned to Laser 1 and Channel FL2. CAUTION For accurate data results, the instrument optics setup must match the current Instrument Configuration. Modifications to the current configuration will not apply unless you click Set Configuration.
2 Press Ctrl-E to create a new Experiment; rename the Experiment DNA.
! Tip
To place the Experiment inside an existing folder, select the folder before creating the Experiment.
3 Rename the new Specimen DNA QC Kit and rename the first Tube CEN.
This Tube will be used to optimize signals from the first laser. Your Experiment should look similar to that shown in the figure at the right.
4 Click the Parameters tab in the Inspector and make the following changes:
Delete all parameters except FSC, SSC, and PI. Select the Height and Width checkboxes for PI. Verify that the Log checkbox is deselected for all parameters.
116
5 Click the Threshold tab and change the threshold parameter to PI; verify
that the threshold value is set to 5,000.
6 Create the following plots for the DNA Tube:

FSC-A vs SSC-A dot plot PI-A vs PI-H dot plot PI-A vs PI-W dot plot PI-A histogram
7 Create a Statistics view and display the mean and CV for PI-A and PI-H.
Right-click the CEN Tube and choose Create Statistics View. Click the Edit Statistics button in the Inspector. On the Population tab, deselect # Events and %Parent. On the Statistics tab, select the Mean and CV for PI-A and PI-H (Figure 5-3 on page 118). Set Decimal Places to 1 for the CVs.
117
Figure 5-3 Setting up the Statistics view
8 In the Acquisition Controls frame, set the Number to Record to 10,000 evt
and the Events to Display to 500 evt.
! Tip
Decreasing the number of displayed events will increase the data refresh rate.
Running CEN
1 Install the CEN sample tube on the cytometer; turn the Fluidics Control
knob to Run.
2 Verify that the green Acquisition pointer is in front of the CEN Tube in the
Browser; click once on the pointer to start acquisition. Events appear in the plots.
3 Adjust the Sample Differential knob to obtain an event rate of

approximately 1,000 events/second. The event rate is displayed in the Acquisition Status frame.
118
4 Adjust the FSC and SSC voltages to place the CEN on scale in the FSC vs
SSC dot plot.
Figure 5-4 FSC and SSC voltages adjusted
5 Adjust the PI voltage to place the singlet nuclei at approximately channel

50 x 103 on the PI-A axis in the PI-A histogram plot.
6 While viewing the PI-A vs PI-H plot, adjust the Y control and the
Excitation Beam Focus wheel to maximize the signals.
Figure 5-5 Optimized PI-A and PI-H signals
7 Decrease the event rate to approximately 200 events/second.
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8 Draw an Interval gate around the first two peaks on the PI-A histogram;
name the populations Singlets and Doublets (Figure 5-6).
CV <3%
Figure 5-6 Defining Singlet and Doublet populations
9 Verify that the FL1/FL2 iris is open and check the CV of the Singlet
population. If the CV is 3%, continue with step 10. If the CV is >3%, skip to the following section; then return to step 10 to finish this section.
10 Click Record to save the data. 11 Check the linearity and print the worksheet.
Note the means of the Singlet and Doublet populations. Divide the mean of the Doublets by the mean of the Singlets. The Doublet/Singlet ratio should be 2.00 +/-0.05. If you cannot achieve a ratio between 1.952.05, contact BD Customer Support. Copy the means, CVs, and the calculated linearity result into the QC log.
12 Remove the CEN sample from the cytometer.
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Optimizing the CV of the Singlet Population

Perform the steps in this section only if the CV of the Singlet population is >3%. After optimizing the CV, return to step 10 in the previous section and finish the remaining steps in that section.
1 Adjust the event rate to approximately 1,000 events/second. 2 Adjust the instrument optimization controls to obtain the lowest possible
CV for the Singlet population. Ensure that the Display is set to 100500 events. Close the FL1/FL2 iris. While viewing the PI-A vs PI-H plot, adjust the Y control, Excitation Beam Focus wheel, X control, Fluorescence focus control knob, Fluorescence channel height adjustment wheel, and FL1/FL2 beam splitter, as necessary.
NOTE PI-stained nuclei are used in this exercise, so the fluorescence signal will be generated by the first laser and collected in the FL2 channel. If you are setting up for an experiment using a different laser and detector channel, peak the signals from that laser using the appropriate rotators, translators, and beam splitters.
3 Decrease the event rate to approximately 200 events/second. 4 Open the FL1/FL2 iris and check the CV of the Singlet population.
If the CV is within an acceptable range, go to step 10 on page 120. If the CV is not within an acceptable range, repeat steps 1 through 4 in this section.
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CTN Resolution
Singlets can be distinguished from aggregates based on size. With BD FACSDiVa software, aggregates can be resolved from singlets on an Area vs Height plot in conjunction with an Area vs Width plot. On the Area vs Height plot singlets can be distinguished from doublets by their height on the y-axis; singlets have slightly more height. On the Area vs Width plot, singlets are distinguished from doublets by the Width measurement; singlets have a smaller Width measurement. Discriminating the singlets from the aggregates enhances the accuracy of cellcycle analysis.
Running CTN
1 Install the CTN sample tube onto the cytometer. 2 Adjust the event rate to approximately 500 events/second. 3 Click the Next button in the Acquisition Controls frame;
change the name of the new Tube to CTN. The Next button duplicates the CEN Tube and Analysis objects. The new plots and Statistics view appear below the previous objects on the worksheet. Acquisition starts automatically and events appear in the plots. Notice the lack of resolution between the singlets and doublets (Figure 5-7).
doublets singlets
Figure 5-7 Unresolved singlets and doublets in unzoomed plot
122
4 Adjust the PI voltage to place the first peak at approximately channel

50 x 103 on the PI-A axis.
5 Focus the laser using the Excitation Beam Focus wheel.

As the laser is focused, the Area measurement will decrease (Figure 5-8).
Figure 5-8 Decrease in Area measurement as laser is focused
6 Adjust Area Scaling.

Click the Laser tab in the Instrument Status frame. Adjust Area Scaling for the first laser until the PI-A intensity is similar to the PI-H intensity.
Figure 5-9 Area Scaling adjusted
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7 Adjust the PI voltage to place the singlets at approximately 50 x 103, if

necessary.
8 (Optional) Use the Zoom-In tool to magnify the area showing the singlets
and doublets on the PI-A vs PI-W plot. Magnify the area from the left of the singlets to the right of the doublets, including only the area of interest. The zoomed-in area should be long and narrow.
Figure 5-10 Doublet discrimination in zoomed-in plot
9 (Optional) Draw a gate around the singlet CTN. 10 Decrease the event rate to approximately 200 events/second. 11 Click Record to save the data; print the worksheet. 12 Remove the CTN Tube from the cytometer; put the instrument in Standby.
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Optimization for Data Recording

Optimize the instrument settings for the actual sample.
1 Install the sample tube and optimize the FSC and SSC signals. 2 Optimize the PI voltage to place the singlets close to channel 50 x 103. 3 Verify the doublet discrimination by zooming in on the PI-A vs PI-W plot.
If the singlets are not resolved, repeat step 6 on page 119. After optimizing the instrument settings, record data for each sample tube. Export data files for analysis in a third-party application such as ModFit LT.
125
126
6
Calcium Flux
The following topics are covered in this chapter: Intracellular Calcium Concentration on page 128 Calcium Flux Optimization on page 129 Measuring Calcium Flux on page 135
127
Intracellular Calcium Concentration

Flow cytometry can be used to measure the concentration of intracellular free calcium ions. Measurement of calcium ion (Ca++) concentration can be made on large numbers of single cells, which provides information about the number of responding cells as well as the relative magnitude of the response to a given stimulus. Ca++ concentration can be correlated with other parameters, such as time, phenotype, and cell cycle. In their resting state, eukaryotic cells maintain an internal Ca++ concentration far less than that of the extracellular environment. Elevation in intracellular Ca++ concentration is often used as an indicator of cellular activation in response to a stimulus. Calcium flux is also an indicator of whether the cells in a population remain functional after exposure to a drug or other compound. Several fluorescent dyes measure intracellular Ca++ levels. For most of them, the amount of Ca++ entering a cell is indicated by a change in fluorescence emission. For example, the emission spectrum of indo-1 changes from blue to violet upon binding to Ca++. The ratio of violet to blue fluorescence is independent of the amount of dye within the cell. When normal cells are analyzed for calcium flux with indo-1 by flow cytometry, a shift in the violet/blue ratio is obtained (Figure 6-1). A break in data occurs when the stimulus is added to the sample tube. The increase in the ratio over time reflects the increase in intracellular Ca++ concentration.
Figure 6-1 Calcium flux data
128
Calcium Flux Optimization

Before beginning this section, do the following: Optimize the instrument electronics as described in Chapter 2, Instrument Setup and Optimization. Ensure the appropriate filters are installed. For the UV1 filter, use 405/20 (violet); for the UV2 filter, use 485/22 (blue); for the beam splitter, use 505 SP. See Appendix A, Optical Configurations, for the layout. Review the following section, Using the Time Parameter.
Using the Time Parameter

The Time parameter can be used to show how events change over time. In calcium flux experiments, the Time parameter is used to display the rate at which the cells in the sample respond to a stimulus. The Time parameter is displayed on a fixed scale of 0262,143, where each tick represents 10 ms. Thus, an event that appears at position 50,000 on the Time scale is equal to 8 min 20 sec; an event that appears at 60,000 is equal to 10 minutes. A plot can display up to 43 minutes of Time data. When you append data to a recorded Tube, time is added to the existing data set. Thus, after appending 5 minutes of data to a 10-minute data set, the Time parameter of the last event would appear at 90,000.
! Tip
To allow enough time for Ca++ flux response and resolution, enter a large value for the Events to record before recording events. You cannot enter a specific time in which to record events or assign a time resolution. Do not restart data recording during a calcium flux experiment.
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129

configuration. Make sure that the configuration lists the UV1 and UV2 parameters, and that the lasers and channels correspond to the optical bench configuration.
! Tip
Add Violet to the UV1 Parameter name and Blue to the UV2 Parameter name to help keep track of UV parameters.
CAUTION For accurate data results, the instrument optics setup must match the current Instrument Configuration.
2 Press Ctrl-E to create a new Experiment; rename the Experiment Calcium

Flux.
! Tip
To place the Experiment inside an existing folder, select the folder before creating the Experiment.
3 Rename the first Tube Ca 1.

This Tube will be used to optimize signals from the UV laser. Your Experiment should look similar to that shown in the figure at the right.
130
4 Click the Parameters tab in the Inspector and make the following changes:
Delete all parameters except FSC, SSC, UV1 Violet, and UV2 Blue. Verify that the Log checkbox is deselected for all parameters.
5 Click the Ratio tab and click the Add button; choose UV1Violet-A for the
Numerator and UV2 Blue-A for the Denominator.
6 Create the following dot plots for the Ca 1 Tube:

FSC-A vs SSC-A UV1Violet-A vs UV2 Blue-A Time vs Ratio: UV1Violet-A/UV2 Blue-A
7 Show the Tube and Population names in the plot titles.

Select all plots on the worksheet, click the Title tab in the Inspector, and select the appropriate checkboxes (Figure 6-2 on page 132).
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Figure 6-2 Showing Tube and Population names in plot titles
8 Create a Statistics view and display the mean for the UV parameters and
the ratio. Right-click the Ca 1 Tube and choose Create Statistics View. Select the Statistics view, and click the Edit Statistics View button in the Inspector. Add the required statistics; delete #Events and %Parent from the Population tab.

1,000,000 evt and the Events to Display to 500 evt.
NOTE Only the specified number of events is displayed in plots during acquisition and recording. After data recording is complete, all recorded events will be displayed.
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Optimizing the Calcium Sample

1 Install the unstimulated sample on the cytometer; turn the Fluidics Control
knob to Run.
2 Verify that the green Acquisition pointer is in front of the Ca 1 Tube in the
Browser; click once on the pointer to start acquisition. Events appear in the plots.
3 Adjust the sample differential to obtain an event rate of approximately

1,000 events/second. The event rate is displayed in the Acquisition Status frame.
4 While viewing the FSC vs SSC plot (Figure 6-3), make the following
adjustments: Adjust the FSC and SSC voltages to place the sample on scale in the FSC vs SSC dot plot. Adjust the FSC threshold to remove debris without cutting into the population of interest. Draw a gate around the lymphocytes; use the Population Hierarchy view to rename the population Lymphocytes.
Figure 6-3 Adjusting FSC and SSC voltages
133
5 Format the remaining two dot plots to show the Lymphocyte population.
Select the two plots, right-click inside one of the plots and choose Show Populations > Lymphocytes.
6 Adjust the UV1 and UV2 voltages to optimize the signal.

The signal should extend along the UV2 axis and should be slightly off the baseline for both axes.
7 Adjust the Ratio Scaling to set the baseline between 050,000.

Select the Tube in the Browser and click the Instr. Settings > Ratio tab in the Inspector. To adjust the setting, select the value in the Scaling field, enter a new value, and press Enter. Repeat as needed to achieve the required results.
134
Measuring Calcium Flux

Do the following to record data for a calcium flux experiment.
1 Change the Events to Display to 50,000 events. 2 Verify that the unstimulated sample is still running; adjust the event rate to
approximately 200 events/second. The event rate is displayed in the Acquisition Status frame.
3 Click Record. 4 When approximately 10,000 events have been recorded, turn the Fluidics
Control knob to Standby and remove the unstimulated sample tube from the cytometer. CAUTION Do not stop recording or acquisition or the data display will reset to zero.
5 Add the stimulus to the tube and mix thoroughly. 6 Reinstall the tube on the cytometer; turn the Fluidics Control knob to Run.
After a few seconds, the Ca++ concentration begins to increase on the Time vs Ratio plot (Figure 6-4).
unstimulated sample
stimulus added
Figure 6-4 Cellular response to stimulus over time
135
7 Click the Acquisition pointer when the cells are no longer reacting to the
stimulus.
8 Remove the tube from the cytometer. 9 Clean the fluidics system with 10% bleach for 5 minutes, and then with
deionized water for 5 minutes. NOTE Make sure to remove any remaining stimulus that would activate cells in subsequent samples.
10 To run another sample, install the next sample tube onto the cytometer. 11 Click the Next button; rename the new Tube as appropriate. 12 Repeat steps 3 through 10.
Analyzing Data
NOTE You can export the data for analysis in a third-party application, such as FlowJo.
1 To better visualize the cellular response, draw a series of Interval gates on

the Time vs Ratio dot plot (Figure 6-5).
Figure 6-5 Calcium flux data points over time
136
2 Use the Statistics view to display the increase in the Ratio mean over time.
137
138
7
Troubleshooting
The tips in this section are provided to help you troubleshoot issues that might arise when using the BD FACSDiVa option. For instrument-specific troubleshooting, refer to the BD FACSVantage SE Users Guide; for softwarespecific troubleshooting, refer to the BD FACSDiVa Software Users Guide. If additional assistance is required, contact your local BD Biosciences technical support representative. See Technical Assistance on page ix. Troubleshooting suggestions in this chapter are grouped under the following headings: Electronics Troubleshooting on page 140 Acquisition Troubleshooting on page 142 Sorting Troubleshooting on page 149
139
Electronics Troubleshooting
Observation Instrument Disconnected in Instrument frame Possible Causes Power switched off on BD FACSDiVa module Communication failure between workstation and instrument Recommended Solutions Check the digital oscilloscope. If the screen is blank, switch on the Digital control switch. Quit the software and then restart it. If restarting does not work, reset the BD FACSDiVa electronics by switching off the power switch on the back of the BD FACSDiVa module, and then switching the power back on. Restart the computer. Unplug and then plug in the cable connectors and make sure they are secure. Enter the correct IP address. Call BD Biosciences for assistance. Wait two minutes. If the message remains, restart the computer. Decrease the event rate or verify the threshold. Delete Analysis objects, decrease the Display value, or delete parameters from the Instrument Settings Inspector.
Ethernet cable disconnected between workstation and instrument IP address changed Upgrading firmware in Instrument frame Master DAQ Overflow in Instrument frame Firmware loading incomplete Event rate too high Too many Analysis objects on worksheet or too many events displayed
140
Electronics Troubleshooting (continued)

Observation Instrument not responding in Status tab Possible Causes Unknown Recommended Solutions Reset the BD FACSDiVa electronics by switching off the power switch on the back of the BD FACSDiVa module, and then switching the power back on. Restart the computer.
NOTE If this occurs during sorting, turn off the deflection plates before resetting the electronics.
Chapter 7: Troubleshooting
141
Acquisition Troubleshooting
Observation No events in plots after clicking Acquire Possible Causes Acquisition pointer not set to current Tube Not in digital mode Viewing plots for a different Tube Incorrect population(s) in plot Recommended Solutions Click to move the Acquisition pointer in front of the appropriate Tube. Switch the Digital control switch to On. Double-click the current Tube in the Browser to display the plots for that Tube. Right-click the plot and choose Show Populations. Verify that the appropriate populations are displayed. Format the plot to display all events. Assign a color to the population displayed in the plot. Verify the population drawing order. Current Instrument Configuration different from optical bench Verify that the current Instrument Configuration corresponds to the optical bench setup. See Instrument Optimization and Quality Control on page 24. Add sample to tube or install new sample tube. Mix sample to suspend cells. Replace the sample tube. Set the threshold to the correct parameter for your application.
Uncolored events in plot
No sample in tube Sample not mixed properly Sample tube cracked Threshold not set to correct parameter (usually FSC)
142
Acquisition Troubleshooting (continued)

Observation No events in plots after clicking Acquire (continued) Possible Causes Multiple Threshold parameters not set correctly Threshold channel too low or too high Unexpected results after clicking Next No fluorescent signal Acquisition pointer on wrong Tube Current Instrument Configuration different from optical bench Recommended Solutions Verify that the correct Boolean logic (And/Or) was used for the Threshold parameters. Adjust the Threshold channel. See Adjusting the Voltages and Threshold on page 56. Verify the Acquisition pointer is in front of the Tube you want to duplicate before clicking Next. Verify that the current Instrument Configuration corresponds to the optical bench setup. See Instrument Optimization and Quality Control on page 24. Make sure the appropriate filter is installed for each fluorochrome. See Appendix A for suggestions. Adjust the laser delay settings. See Optimizing Signals from the Second-Laser Intercept on page 37 or Optimizing Signals from the Third-Laser Intercept on page 44. Adjust Area Scaling for the corresponding laser.
Wrong filter installed
Laser delay set incorrectly
Low Area signal
Area Scaling Factor too low
143

Observation Unexpected events in plot Possible Causes Incorrect logic in Population Hierarchy Incorrect population(s) in plot Recommended Solutions Verify the gating strategy. Right-click the plot and choose Show Populations. Verify that the appropriate populations are displayed. Verify that the required population is not hidden by another population. Right-click the plot and choose Order Populations by Count. Adjust the Threshold channel. See Adjusting the Voltages and Threshold on page 56. Dilute the sample. Decrease the event rate using the Sample Differential knob. Remove the air bubble. Refer to the BD FACSVantage SE Users Guide. Turn off test signals from the instrument control panel. Put the instrument in Standby and adjust the FSC obscuration bar to remove the noise. Decrease the Drop Drive amplitude.
Incorrect drawing order
Unexpectedly high event rate
Threshold channel too low
Sample too concentrated Event rate too high Air bubble
Test signal interference Laser noise
144

Observation Unexpectedly low event rate Possible Causes Memory full Recommended Solutions Compare the processed event rate in BD FACSDiVa software with the threshold counter on the instrument. If the BD FACSDiVa event rate is much lower, quit and then restart the application. Adjust the Threshold channel. See Adjusting the Voltages and Threshold on page 56. Mix the sample to suspend cells. Concentrate the sample. Filter the sample. Replace the sample tube. Replace the O-ring. Refer to the BD FACSVantage SE Users Guide. Clear the nozzle tip as described in the BD FACSVantage SE Users Guide. Re-stain the sample, making sure the tube is clean. Fill the sheath tank.
Threshold channel too high
Sample not adequately mixed Sample too dilute Erratic event rate Sample aggregates Sample tube cracked Sample tube O-ring worn
Nozzle tip clogged
Sample contaminated Sheath tank low
145

Observation Distorted scatter parameters Possible Causes Instrument settings adjusted incorrectly Air bubble Recommended Solutions Optimize the scatter parameters. See Adjusting the Voltages and Threshold on page 56. Remove the air bubble. Refer to the BD FACSVantage SE Users Guide. Clean the nozzle tip as described in the BD FACSVantage SE Users Guide. Increase the Threshold channel. See Adjusting the Voltages and Threshold on page 56. Examine the sample under a microscope. Re-stain the sample, making sure the tube is clean. Verify the instrument alignment. Decrease the event rate using the Sample Differential knob. Repeat sample preparation. Make new QC samples and perform the quality control procedure again. Increase the Window Extension.
Nozzle tip clogged or dirty
Excessive amount of debris in plots
Threshold channel too low
Dead cells or debris in sample Sample contaminated High CVs Instrument not aligned Event rate too high Poor sample preparation Old or contaminated quality control (QC) particles Window Extension too low
146

Observation High electronic abort rate (>10% of system event rate) Possible Causes Event rate too high Sample aggregated Sample too concentrated Threshold channel too low Window Extension too high Fewer events than expected in gated population Events left out of gate Plot zoomed Laser delay set incorrectly Recommended Solutions Decrease the event rate. Filter the sample. Dilute the sample. Increase the threshold channel. Decrease the Window Extension. When drawing a gate, make sure events on the axis are included. Unzoom the plot or make the gate bigger. Adjust the laser delay settings. See Optimizing Signals from the Second-Laser Intercept on page 37 or Optimizing Signals from the Third-Laser Intercept on page 44. Adjust the Window Extension. Refer to the BD FACSDiVa Software Users Guide, if needed. Slightly increase the Window Extension to maximize Area signal.
Window Extension set incorrectly Increasing threshold results in decreased Area signal Window Extension too small
CAUTION Increasing the Window Extension too much results in more electronic aborts or high CVs.
147

Observation Area measurement offscale while the Height measurement is on scale Possible Causes Area Scaling factor too high Recommended Solutions Decrease the Area Scaling factor to move the Area measurement back on scale. If necessary, adjust Area Scaling to make the Area measurement match the Height measurement. Select the row using the selection button.
Cannot delete from Inspector
Row not selected
148
Sorting Troubleshooting
Observation Function keys not responding for Sort Setup values Defined population not listed in Add menu Unusual pattern on digital oscilloscope while setting Phase Sort button disabled Possible Causes Sort Setup frame inactive Recommended Solutions Select the frame title bar to make it active and then press the required function key. Redefine the population using another gate type. Turn on Test Sort, and then push the AUTOSET button on the oscilloscope control panel. Click to move the Acquisition pointer in front of the appropriate Tube. Double-click the appropriate Tube in the Browser to view worksheet objects for that Tube. Decrease the event rate. Verify the gating hierarchy.
Population defined using Snap-To gate Test Sort off
Acquisition pointer not set to current Tube Viewing Sort Layout for another Tube Event rate too high for Drop Drive frequency Sorting parent and child populations into two different tubes Purity Mask too high
Sort Layout counters not updating High sort conflict rate
Decrease the Purity Mask. Use the arrow buttons to choose the appropriate stream. See Using Sorting Controls on page 81. Decrease the Drop Drive amplitude. Decrease the event rate.
Unexpected sort rate
Wrong stream displayed in Sorting status bar Laser noise
Erratic sort rate
Event rate too high
149
Sorting Troubleshooting (continued)

Observation Unexpected sort results Possible Causes Incorrect logic in Population Hierarchy Sorting parent and child populations into two different tubes Recommended Solutions Verify the gating strategy. If you try to sort a parent and its child population into two tubes, BD FACSDiVa software ignores the child events in both tubes. Create a new subset under the parent population consisting of NOT (Child). Sort the child population into one tube and the NOT (Child) population into another tube. Unable to move stage in Status tab Electronics mode set incorrectly BD QuadraSort tube holder installed No stream when sorting onto a plate or slide Insufficient stream deflection for four-way sort Value for wrong stream entered in Streams tab Deflection angle adjusted incorrectly Verify that the instrument is in digital mode. Remove the BD QuadraSort hardware. Enter a value for the Far Left stream. Increase the plate voltage. Change the angles of the tube holders. Lower the position of the BD QuadraSort tube holder. Place a spacer in the outer tube holders to raise the level of the outer tubes. Cracked tubes with BD QuadraSort 15-mL tube holder Wrong tubes used Use only polypropylene tubes (BD Catalog No. 352096) with the BD QuadraSort tube holder.
150
Appendix A Optical Configurations

The diagrams in this appendix show how to set up the optical bench to match the default Instrument Configurations. Use the following examples as a guide when setting up your own instrument configuration: Six-Color Configuration on page 152 Alternate Six-Color Configuration: Five Colors + DNA on page 153 Seven-Color Configuration on page 154 Eight-Color Configuration on page 155 Alternate Eight-Color Configuration on page 156
This appendix also includes a blank configuration worksheet that can be photocopied and filled in for any custom configurations. See Configuration Worksheet on page 157.
151
Six-Color Configuration
FITC (FL1) 575/26 PE (FL2) 530/30 560 SP
712/21 610 SP 405/20 UV1 (FL4) 505 SP 485/22 SSC UV2 (FL5) APC (FL6) 660/20 PerCP-Cy5-5 (FL3)
152
Alternate Six-Color Configuration: Five Colors + DNA
FITC (FL1) 575/26 PE (FL2) 530/30 560 SP
712/12 610 SP 424/22 Hoechst 33258 (FL4) 780/60 APC-Cy7 (FL6) 710 LP 660/20 SSC APC (FL7) PerCP-Cy5-5 (FL3)
Appendix A: Optical Configurations
153
Seven-Color Configuration
FITC (FL1) 575/26 PE (FL2) 530/30 560 SP
712/21 610 SP 405/20 UV1 (FL4) 505 SP 485/22 SSC UV2 (FL5) APC (FL7) 780/60 APC-Cy7 (FL6) 710 LP 660/20 PerCP-Cy5-5 (FL3)
154
Eight-Color Configuration
FITC (FL1) 575/26 PE (FL2) 530/30 560 SP PE-Cy5 (FL8) 710/20 740 LP 750 LP or 780/60 PE-Cy7 (FL3) 780/60 APC-Cy7 (FL6) 710 LP 660/20 UV2 (FL5) SSC APC (FL7)
610 SP 405/20 UV1 (FL4) 505 SP 485/22
155
Alternate Eight-Color Configuration*
FITC (FL1) 575/26 PE (FL2) 530/30 560 SP PE-Tx Red (FL8) 610/20 640 LP 740 LP or 780/60 PE-Cy7 (FL3) 780/60 or 740 LP APC-Cy7 (FL6) 710 LP 660/20 Hoechst Red (FL5) APC (FL7)
610 SP 450/20 Hoechst Blue (FL4) 610 SP 675 LP SSC
* For more information, see Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. Goodell MA, Brose K, Paradis G, et al. J Exp Med; 1996:183;1797-1806.
156
Configuration Worksheet
Configuration Name: ________________________________
FL ____ =
FL ____ =
FL ____ =
FL ____ =
FL ____ =
FL ____ =
FL ____ = SSC
FL ____ =
157
158
Index
A
aborts electronic 147 See also conflicts, sort. 80 AccuDrop determining drop delay 102 Experiment 100 monitor 15, 100 acquisition events to record 62 starting 31 troubleshooting 142 Acquisition Templates creating 61 previewing data 61, 67 adjusting area scaling 33, 41, 47, 123 Home location 71, 108 laser delay 40, 46 sort settings 72, 95 streams 31, 75 threshold 56 voltages 56 Window Extension 40, 46 alignment sample, preparing 24 Alpha control 30 amplitude about 74 optimizing 96 analog data (vs digital) 19 operation 17 oscilloscope 15, 32 analysis calcium flux data 136 data 61 DNA data 125 immunophenotyping 64 primary-laser results 35 reusing 67 saving 67 second-laser results 43 third-laser results 49 applications calcium flux 128 DNA 114 sorting 90 area scaling adjusting 41, 47 DNA experiment 123 primary laser 33 troubleshooting 143, 148 assistance, technical ix Attenuation 73
159
B
Backspace key, troubleshooting ix beads AccuDrop 100 alignment 24 sorting for drop delay 102 biohazardous hardware 90 waste 22 breakoff about 74 adjusting settings 95 adjusting with keyboard 72, 96 controls 74 optimal distance 96
C
calcium flux about 128 data analysis 136 Experiment 130 measuring 135 optimization 129 optimizing sample 133 Time parameter 129 calculating compensation 59, 60 drop delay 100 calf thymocyte nuclei (CTN) preparing 115 resolution 122 running 122 CellQuest Pro, viewing 18 chicken erythrocyte nuclei (CEN) Experiment 116 optimization 115 preparing 115 running 118 coefficient of variation (CV), high 146
compensation calculating 59, 60 gating data 59 Tubes, creating 55 components AccuDrop monitor 15 digital oscilloscope 14 FACSDiVa module 14 QuadraSort 17 computer starting up 23 workstation 18 configurations alternate eight-color 156 eight-color 155 five colors + DNA 153 instrument defaults 151 seven-color 154 six-color 152 variants 37 worksheet 157 conflicts, sort about 84 counting 82 printing 83 saving 80 troubleshooting 149 control switch about 15 analog operation 17 digital operation 15 controls Breakoff controls 74 compensation 55 inactive 16 instrument 16, 30, 70 single-stained 53 sort setup 71 sorting 70, 81 Streams controls 75
160
conventions viii counters, sort 82, 149 creating Acquisition Templates 61 Analysis objects 64 compensation Tubes 55 custom devices 71, 110 Sort Layouts 70, 79, 104 Sort Precision modes 70, 89 custom devices creating 71, 110 deleting 111 customer support ix cytometer See instrument.
DNA experiments, about 114 five colors + configuration 153 QC Particles kit 115 setting up Experiment 116 verifying linearity 114 doublet discrimination 114, 120, 124 drop charge 75 conflicts 84 correction factor 75 delay 74, 100 drive 73 sequence 74 starting values 98
D
data analyzing 61, 64 digital vs analog 19 gating 59, 64 recording 61, 63 delay drop 100 laser 40, 46 Delete key, troubleshooting ix deleting custom devices 111 sort populations 81 sort setup values 76 digital control switch 15 data (vs analog) 19 oscilloscope 14 digital mode active controls 70 inactive controls 16 operation 15 starting instrument in 22
E
editing Sort Layouts 81 eight-color configuration 155, 156 electronics aborts 147 about 12 control switch 15 troubleshooting 140 error messages instrument disconnected 140 instrument not responding 141 Master DAQ overflow 140 Unable to move stage 150 upgrading firmware 140 events not showing in plots 142, 144 rate, troubleshooting 144, 145 troubleshooting 147 Excitation Beam Focus wheel 30
Index
161
Experiments AccuDrop optimization 100 calcium flux 130 CEN optimization 116 immunophenotyping 61 instrument optimization 25 reusing 50 sample optimization 54 exporting Sort Reports 84
H
hardware biohazardous 90 plate sorting, installing 107 QuadraSort, installing 93 two-way sorting, installing 93 Home Device 71 Home location adjusting 71, 108 custom devices 110
F
FACSDiVa option about 12 components 13 workstation 18 FACStation 18 Far Left stream 75 Far Right stream 75 Fine Tune mode 89 fluorescence channel height adjustment wheel 30 focus control knob 30 signal, troubleshooting 143 frequency about 74 optimal ranges 96 function keys, troubleshooting 72, 149
I
immunophenotyping analysis 64 Experiment 61 Initial mode 89 Inspector, troubleshooting 148 installing nozzle tips 90, 92 plate-sorting hardware 107 QuadraSort hardware 93 two-way sorting hardware 93 instrument controls 16, 30, 70 default configurations 151 disconnect error 140 not responding 141 optimization 24 starting up 22 intracellular calcium concentration 128
G
gating compensation Tubes data 64 59
K
keyboard shortcuts 72 troubleshooting
ix, 72, 149
162
L
labels parameter 62 lasers delay 40, 46 primary, optimizing signals 30, 32 QC results 35, 43, 49 second, optimizing signals 37, 39 third, optimizing signals 44, 45 Layout, Sort See Sort Layouts. Left stream 75 linearity, DNA experiments 114 low Area signal 143
O
obscuration bar 30 on/off switch about 15 analog operation 17 digital operation 15 optical configurations 151 optimization AccuDrop 100 CEN 115 CV of singlet population 121 for calcium samples 129, 133 for DNA samples 114, 125 for LWB samples 53 instrument 24, 25 primary-laser signal 30, 32 sample 52 second-laser signal 37, 39 third-laser signal 44, 45 oscilloscope analog 15, 32 digital 14 troubleshooting 149
M
main sorting adjustments 92 Masks about 84 default Precision Modes 88 Phase 87 Purity 86 Yield 85 Master DAQ overflow error 140 measuring calcium flux 135 modes defining 89 Sort Precision 84, 88 monitoring sorts 83, 106 monitors AccuDrop 15, 100 computer 18
P
parameters labels 62 scatter, distorted 146 Time 129 pausing sorting 81, 106 phase about 74 optimizing 97 Phase Masks about 87 Yield Masks, using with plates, sorting into 106
N
nozzle tips installing 90, 92 optimal frequency with 96 sizes and catalog numbers 91
86, 88
Index
163
plots excessive debris 146 no events in 142 unexpected events in 144 populations AccuDrop bead, defining 100 sorting 79, 91, 104 troubleshooting 147, 149 Precision Modes 84, 88 See also Sort Precision modes. primary laser optimizing signals 30, 32 results 35 verifying area scaling 33 printing Sort Reports 84, 99 Purity Masks 86 Purity mode 88
S
sample alignment 24 tubes, QuadraSort 93, 94 sample optimization about 52 Experiment 54 LWB example 53 samples, running 63 Save Conflicts 80 saving analyses 67 sort conflicts 80 sort setup values 76, 99 scaling, area DNA experiment 123 primary laser 33 second laser 41 third laser 47 troubleshooting 143, 148 scatter parameters, distorted 146 second laser adjusting delay 40 optimizing signals 37, 39 results 43 verifying area scaling 41 seven-color configuration 154 sheath filling tank 22 pressures, optimal frequency with 96 shortcuts, keyboard 72 signals low Area 143 no fluorescent 143 primary laser, optimizing 30, 32 second laser, optimizing 37, 39 third laser, optimizing 44, 45 troubleshooting 148 Single Cell mode 89 single-stained controls 53
Q
QuadraSort about 17 installing hardware 93 tube placement 94 quality control 24
R
recording calcium flux data 135 compensation Tubes 58, 59 data 61, 63 DNA data 125 primary-laser results 35 second-laser results 43 third-laser results 49 reports, sort 83 results, troubleshooting 143, 150 reusing analyses 67 Right stream 75
164
singlet population defining 120 optimizing CV 121 six-color configuration 152 slides, sorting into 106 software about 12 starting up 23 Sort Layouts about 77 creating 70, 79, 104 custom 71, 110 editing 81 entering populations 79 examples 77 Sort Precision modes about 84 creating 70, 89 defaults 88 Fine Tune 89 Initial 89 Purity 88 Single Cell 89 Yield 88 sort rate, troubleshooting 149 Sort Reports displaying 71, 83 exporting 84 printing 84, 99 Sort Setup controls about 71 Breakoff controls 74 button functions 73 Streams controls 75 sorting about 69, 90 adjusting settings 72, 95 beads for drop delay 102 collection devices 77 conflicts 80, 82, 84
controls 70, 81 counters 82 into plates 106 into slides 106 into tubes 92 main adjustments 92 monitoring 83, 106 pausing 81, 106 populations 79, 91, 104 report 83 saving settings 76, 99 setting up for 90 Sort button 81 starting 81, 104 stopping 81 test mode 73 troubleshooting 149 starting acquisition 31 computers 23 instrument 22 sample flow 31 software 23 sorting 81, 104 stopping sorting 81, 104 streams adjusting 31, 70, 75 Streams tab 75 troubleshooting 150 turning on 73
T
tanks, servicing 22 Target Events 79 technical assistance ix Test Sort 73 test tubes, sorting into 92 Theta control 30
Index
165
third laser adjusting delay 46 optimizing signals 44, 45 results 49 verifying area scaling 47 threshold adjusting 56 troubleshooting 147 Time parameter 129 troubleshooting acquisition 142 CVs 146 electronic aborts 147 electronics 140 event rate 144, 145 Inspector 148 keyboard keys ix, 72, 149 low Area signal 143 oscilloscope 149 plots 142, 144, 146 populations 147, 149 results 150 scatter parameters 146 signals 143, 148 sort conflict rate 149 sort counters 149 sort rate 149 sorting 149 streams 150 tubes 150 Window Extension 147 Tubes compensation 55 tubes installing two-tube holder 93 sorting into 92 troubleshooting 150 used with QuadraSort 93, 94
two-way sorting hardware 93 typographical conventions viii
U
upgrading firmware error 140
V
viewing CellQuest Pro voltages, PMT adjusting 56 18
W
waste tank, emptying 22 Window Extension adjusting 40, 46 troubleshooting 147 worksheet, configuration 157 workspace, view options 18 workstation, about 18
X
X control 30
Y
Y control 30 Yield Masks about 85 Phase Masks, using with 86, 88 Yield mode 88
Z
Z control 30 Z distance 31
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http://www.bdbiosciences.com/ Part No. 334556 Rev. A November 2002

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