BD FACSAria User Manual

BD FACSAria

User’s Guide

bdbiosciences.com

Part No. 643632 Rev. A
December 2007

BD Biosciences

San Jose, CA 95131-1807
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© 2007 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 Becton, Dickinson and Company.

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, BD Logo and all other trademarks are property of Becton, Dickinson and Company. © 2007 BD

Clorox is a registered trademark of The Clorox Company. Fluoresbrite is a registered trademark of Polysciences, Inc.
JDS Uniphase is a trademark of the JDS Uniphase Corporation. Microsoft, Windows, and Excel are registered
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trademarks of the respective companies with which they are associated.

Cy™ dyes are subject to proprietary rights of Amersham Biosciences Corp and Carnegie Mellon University and are made
and sold under license from Amersham Biosciences Corp only for research and in vitro diagnostic use. Any other use
requires a commercial sublicense from Amersham Biosciences Corp, 800 Centennial Avenue, Piscataway, NJ 08855­1327, USA.

FCC Information

WARNING: Changes or modifications to this unit not expressly approved by the party responsible for compliance could
void the user’s authority to operate the equipment.

NOTE: This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part
15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the
equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency
energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio
communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case
the user will be required to correct the interference at his or her own expense.

Shielded cables must be used with this unit to ensure compliance with the Class A FCC limits.

This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulations.

Cet appareil numérique de la classe A respecte toutes les exigences du Réglement sur the matériel brouilleur du Canada.

History

Revision Date Change Made

333608 1/03 Initial release

338645 10/04 Updated for new instrument and software features: bubble and ND filters,

640760 5/06 Updated for BD FACSDiva software version 5.0 and new ceramic nozzles

642226 6/07 Updated for BD FACSDiva software version 6.0.

643632 12/07 Updated for BD FACSDiva software version 6.1.

adjustable nozzle, waste cap with baffle and disposable filtered cap, adjustable
nozzle, running samples using BD FACSDiva software version 4.1

Contents

About This Guide ix

Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Safety and Limitations xiii

Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

Laser Product Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

Precautions for Safe Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Biological Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

General Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Precaution Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xviii

Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xx

Chapter 1: BD FACSAria Cytometer Components 21

Fluidics Cart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Containers and Connectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Power and Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Fluidics Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Optics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Cytometer Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

iii

Chapter 2: Theory of Operation 45

Fluid Movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Sheath Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Sample Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Signal Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Fluorescent Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Electronic Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Pulse Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Laser Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Drop Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Side Stream Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Drop Charging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Conflict Resolution During Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Chapter 3: Using BD FACSDiva Software 79

Workspace Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Cytometer Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Fluidics Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Fluidics Level Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Cytometer Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Cytometer Status Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Acquisition Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

iv BD FACSAria User’s Guide

Sorting Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Sort Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Sort Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Sort Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Sort Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Chapter 4: Running Samples 107

Cytometer Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Setting Up the Breakoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Setting Up the Fluidics Cart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Checking Performance with Cytometer Setup and Tracking . . . . . . . . . . . . 118

Overview of Cytometer Setup and Tracking . . . . . . . . . . . . . . . . . . . . . . 118

Cytometer Quality Control Using BD FACSDiva Software . . . . . . . . . . . . . 119

Setting Up Cytometer Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Preparing QC Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Adjusting Area Scaling and Laser Delay . . . . . . . . . . . . . . . . . . . . . . . . . 122

Reusing the QC Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Tracking QC Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Setting Up the Workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Calculating Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Data Recording and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Setting Up the Global Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Recording Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Performing Batch Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Sample QC Log . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Contents v

Chapter 5: Sorting 153

Setting Up for Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Setting Up for Bulk Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Determining the Drop Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Sorting Beads to Determine the Drop Delay . . . . . . . . . . . . . . . . . . . . . . 159

Determining the Drop Delay - Automatic Method . . . . . . . . . . . . . . . . . . . . 162

Overview of Auto Drop Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Using Auto Drop Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Setting Up the Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

Starting and Monitoring the Sort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Setting Up for Sorting Into a Plate or Slide . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Installing the Sorting Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Setting Up the Stream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Creating a Custom Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Chapter 6: Shutdown and Maintenance 177

Daily Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Fluidics Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

External Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Scheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Internal Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

Purging Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

Changing Fluid Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Changing the Sample Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Changing the Air Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Changing the Bubble Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Unscheduled Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

vi BD FACSAria User’s Guide

Changing the Nozzle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

Adjusting the Nozzle Orifice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Cleaning the Nozzle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Cleaning the Camera Windows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Cleaning the Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

Purging the Bubble Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214

Chapter 7: Troubleshooting 219

Troubleshooting the Stream . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

Troubleshooting the Breakoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Sorting Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Acquisition Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Fluidics Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

Electronics Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

Appendix A: Supplies and Options 239

Cytometer Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

Optical Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

Accessory Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

Other Replacement Parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244

Laser Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Cytometer Setup Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Labware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

Cytometer Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Application Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

Two-Laser System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

Three-Laser System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Appendix B: Application Settings 257

Contents vii

Application Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Creating Application Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

Index 265

viii BD FACSAria User’s Guide

About This Guide

This user’s guide contains the instructions necessary to operate and maintain
your BD FACSAria™ flow cytometer. Because many instrument functions are
controlled by BD FACSDiva™ software, this guide also contains basic software
information needed for instrument setup. To familiarize yourself with the
software, do the tutorials in the Getting Started with BD FACSDiva Software
guide. For detailed information on software features, refer to the BD FACSDiva
Software Reference Manual.

The BD FACSAria User’s 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.

New users of the BD FACSAria flow cytometer should read:

• Chapter 1 to become familiar with instrument components

• Chapter 2 to understand how the instrument works and to learn about the
software components used to control different subsystems

• Chapter 3 to see where software components are located

Instructions for routine acquisition, analysis, and sorting can be found in
Chapters 4 and 5.

Once you become familiar with routine operation and need only a quick
reminder of the main steps, use the quick reference guide provided with this
user’s guide.

ix

Conventions

The following tables list conventions used throughout this guide. Table 1 lists the
symbols that are used in this guide or on safety labels to alert you to a potential
hazard. Text and keyboard conventions are shown in Table 2.

Table 1 Hazard symbols

Symbol Meaning

Caution: hazard or unsafe practice that could result in material damage, data
loss, minor or severe injury, or death

Electrical danger

Laser radiation

Biological risk

a. Although these symbols appear in color on the instrument, they are in black and white throughout this user’s

guide; their meaning remains unchanged.

Table 2 Text and keyboard conventions

Convention Use

 Tip

a

Highlights features or hints that can save time and prevent
difficulties

NOTICE Describes important features or instructions

Italics Italics are used to highlight book titles and new or unfamiliar

terms on their first appearance in the text.

>

Ctrl-X When used with key names, a dash means to press two keys

The arrow indicates a menu choice. For example, “choose

> Print” means to choose Print from the File menu.

File

simultaneously. For example, Ctrl-P means to hold down the
Control key while pressing the letter p.

x BD FACSAria User’s Guide

Technical Assistance

For technical questions or assistance in solving a problem:

• Read the section of the user’s guide specific to the operation you are
performing.

• 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 system performance

For instrument support from within the US, call [phone].

For support from within Canada, call [phone].

Customers outside the US and Canada, contact your local BD representative or
distributor.

About This Guide xi

THIS PAGE INTENTIONALLY LEFT BLANK

Safety and Limitations

The BD FACSAria flow cytometer is equipped with safety features for your
protection. Operate only as directed in the user’s guide. Do not perform
instrument maintenance or service except as specifically stated. Keep this safety
information available for reference.

Laser Safety

Lasers or laser systems emit intense, coherent electromagnetic radiation that has
the potential of causing irreparable damage to human skin and eyes. The main
hazard of laser radiation is direct or indirect exposure of the eye to thermal
radiation from the visible and near-infrared spectral regions (325–1,400 nm).
Direct eye contact can cause corneal burns, retinal burns, or both, and possible
blindness.

There are other potentially serious hazards in other spectral regions. Excessive
ultraviolet exposure produces an intolerance to light (photophobia) accompanied
by redness, a tearing discharge from the mucous membrane lining the inner
surface of the eyelid (conjunctiva), shedding of the corneal cell layer surface
(exfoliation), and stromal haze. These symptoms are associated with
photokeratitis, otherwise known as snow blindness or welder’s flash, which
results from radiant energy–induced damage to the outer epidermal cell layer of
the cornea. These effects can be the result of laser exposure lasting only a fraction
of a second.

xiii

Laser Product Classification

Laser hazard levels depend on laser energy content and the wavelengths used. A
numbered system is used to categorize laser products according to different
hazard levels. The higher the classification number, the greater the potential
hazard. The BD FACSAria flow cytometer is a Class I (1) laser product per 21
CFR Subchapter J and IEC/EN 60825-1:1994 + A2:2001. The lasers and the
laser energy are fully contained within the instrument structure and call for no
special work area safety requirements except during service procedures. These
procedures are to be carried out only by BD Biosciences service personnel.

Precautions for Safe Operation

Modification or removal of the optics covers or laser shielding could result
in exposure to hazardous laser radiation. To prevent irreparable damage to
human skin and eyes, do not remove the optics covers or laser shielding,
adjust controls, or attempt to service the instrument any place where laser
warning labels are attached (see Precaution Labels on page xviii).

Use of controls or adjustments or performance of procedures other than
those specified in the user’s guide may result in hazardous radiation
exposure.

Keep all instrument doors closed during instrument operation. When
operated under these conditions, the instrument poses no danger of
exposure to hazardous laser radiation.

xiv BD FACSAria User’s Guide

Electrical Safety

Lethal electrical hazards are present in some lasers, particularly in laser
power supplies. Many portions of the electrical system, including the
printed circuit boards, are at a dangerous voltage level. To prevent shock
injury, follow these guidelines.

• Turn off the power switch and unplug the power cord before servicing the
instrument, unless otherwise noted.

• Connect the equipment only to an approved power source. Do not use
extension cords. If cords, plugs, or cables are damaged, immediately
contact BD Biosciences for a replacement.

• Do not remove the grounding prong from the power plug. Have a qualified
electrician replace any ungrounded receptacles with properly grounded
receptacles in accordance with the local electrical code.

• For installation outside the US, use the proper power cord. Contact your
local BD representative for more information.

Do not plug the fluidics cart power cord into a wall outlet. Plug the cord
into the cytometer only. This ensures proper electrical grounding and
protects against electrical shock or damage to the instrument.

A 12,000-volt potential exists between the deflection plates when they are
on. Contact with the charged plates results in serious electrical shock. Do
not touch the deflection plates when the voltage warning light is
illuminated, or when the software indicates that the plate voltage is on. The
plates remain energized even when the sort block door is opened.

The barb and the stream-charging wire conduct electrical charge during
sorting. To prevent shock, do not touch the barb or the stream-charging
wire when the voltage warning light is illuminated or any time during
sorting.

Safety and Limitations xv

Biological Safety

All biological specimens and materials coming into contact with them can
transmit potentially fatal disease. To prevent exposure to biohazardous
agents, follow these guidelines.

• Handle all biological specimens and materials as if capable of transmitting
infection. Dispose of waste using proper precautions and in accordance
with local regulations. Never pipette by mouth. Wear suitable protective
clothing, eyewear, and gloves.

• 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.

• Prevent waste overflow by emptying the waste container daily and
whenever the waste indicator shows the waste is getting full.

• The waste tank can become pressurized when the cytometer is running.
Always disconnect the tank from the wet cart before you empty it. Wait at
least 1 minute for pressure to dissipate before you remove the waste cap or
sensor.

• Replace the waste tank cap every month. Failure to do so could cause the
waste tank to malfunction. For tracking, write the date on the waste cap
label each time you change the cap.

• Do not wet the waste tank cap. If wet, the filter
in the cap will cause the tank to malfunction.
To keep the cap dry, place it on the bench label­side up when it is not on the tank. If you see
liquid in the waste cap trap, remove the drain
plug and fully drain the liquid before you
replace the plug.

xvi BD FACSAria User’s Guide

trap

drain plug

Cell sorters that use droplet generation methods, like the BD FACSAria,
can produce aerosols around the sample stream. When acquiring
biohazardous samples, follow universal precautions at all times. Keep the
sort block door closed during sorting. If you need to access the sort block,
turn off the stream before opening the door.

For information on laboratory safety, refer to the following guidelines.

• Schmid I, Nicholson JKA, Giorgi JV, et al. Biosafety guidelines for sorting

of unfixed cells. Cytometry. 1997; 28:99-117.

• Protection of Laboratory Workers From Occupationally Acquired
Infections; Approved Guideline – Third Edition. Wayne, PA: Clinical and
Laboratory Standards Institute, 2005. CLSI document M29-A3.

• Procedures for the Handling and Processing of Blood Specimens; Approved
Guideline – Third Edition. Wayne, PA: Clinical and Laboratory Standards
Institute; 2004. CLSI document H18-A3.

The two CLSI documents listed above can be ordered online at
www.clsi.org.

• European Council Directive on the protection of workers from risks related
to exposure to biological agents at work (90/679/EEC) and its relevant
national transpositions.

General Safety

Movement of mechanical parts within the instrument can pinch or injure
your hands or fingers. To prevent injury by moving parts, follow these
precautions.

• Keep your hands and clothing away from the sample injection chamber
when a tube is loading or unloading. Do not place objects underneath the
sample injection chamber.

Safety and Limitations xvii

• Keep your hands away from the sort aspirator drawer during sorting. The
drawer can close at any time without warning if a clog is detected during
sorting.

• Keep the sort collection chamber door closed when sorting into a plate. Do
not place objects inside the sort collection chamber, and do not attempt to
access the plate until sorting is complete. The microtiter tray stage can
move suddenly during the sorting process. Sorting continues even when the
sort collection chamber door is open.

Precaution Labels

The following precaution labels appear on the BD FACSAria flow cytometer or
fluidics cart to indicate a potential hazard. Do not remove these labels. Use
appropriate precautions to avoid injury by the indicated hazard. See the previous
sections for more information.

Label Location(s) Potential Hazard

*

Waste

Waste (A)

338677 Rev A

Ethanol



xviii BD FACSAria User’s Guide

Waste tank, waste tank
connectors on fluidics cart

Waste tank cap Risk of exposure to

Ethanol tank, ethanol tank
connectors on fluidics cart

Risk of exposure to
biologically transmissible
disease

biologically transmissible
disease

Highly flammable material

Label Location(s) Potential Hazard



Ethanol

Ꮩ

CAUTION

VISIBLE AND/OR INVISIBLE

CLASS 3B LASER RADIATION

WHEN OPEN. AVOID

EXPOSURE TO THE BEAM.

CAUTION

VISIBLE AND/OR INVISIBLE

CLASS 3B LASER RADIATION

WHEN OPEN AND INTERLOCKS

DEFEATED. AVOID EXPOSURE

TO THE BEAM.

Ꮨ

Deflection

Plates

335749

Ethanol tank cap Highly flammable material

On or near all removable
covers and any place
where the laser beam can
emerge from the
instrument

335751

Sort block door and
deflection plates warning
light

Risk of exposure to
hazardous laser radiation

Dangerous voltage

Behind sample injection
chamber

Risk of crushing or
pinching by moving parts

!

Safety and Limitations xix

Label Location(s) Potential Hazard

Floor of sort collection

CAUTION:

MOVING PARTS

!

chamber

Power input on fluidics

!

WARNING: Before applying
power, configure the power
input module to the supply
voltage. Consult the user's
guide for instructions.

a. NOTE: Voltage is set by your service engineer during installation.

Peel Here

cart

Risk of crushing or
pinching by moving parts

Risk of instrument damage
if wrong voltage applied

Limitations

This instrument is for Research Use Only. Not for use in diagnostic or
therapeutic procedures.

BD Biosciences is providing software without warranty of any kind on an as-is
basis. The software and workstations are intended for running the instruments
supplied by BD Biosciences. It is the responsibility of the buyer/user to ensure
that all added electronic files including software and transport media are virus
free. If the workstation is used for Internet access or purposes other than those
specified by BD Biosciences, it is the buyer/user’s responsibility to install and
maintain up-to-date virus protection software. BD Biosciences does not make any
warranty with respect to the workstation remaining virus free after installation.
BD Biosciences is not liable for any claims related to or resulting from the buyer/
user's failure to install and maintain virus protection.

a

xx BD FACSAria User’s Guide

1

BD FACSAria

Cytometer Components

The BD FACSAria flow cytometer is the first fixed-alignment benchtop high­speed cell sorter. The cytometer can be operated at varied pressures and can
acquire up to 70,000 events per second. With its fixed-optics design and digital
electronics, the BD FACSAria flow cytometer enables multicolor analysis of up to
13 fluorescent markers and two scatter parameters at a time.

The BD FACSAria system consists of three major components: a fluidics cart, a
benchtop flow cytometer, and a workstation (see Figure 1-1 on page 22). Nearly
all cytometer functions are operated from within BD FACSDiva software.

For a description of the BD FACSAria system components, see the following
sections. For technical information about how the cytometer works, see
Chapter 2.

• Fluidics Cart on page 22

• Flow Cytometer on page 26

• Workstation on page 44

21

Figure 1-1 BD FACSAria cytometer components

flow cytometer

fluidics cart

workstation

Fluidics Cart

A separate fluidics cart supplies sheath and cleaning fluids and collects waste
from the cytometer (Figure 1-2). The self-contained fluidics cart supplies the
required air pressure and vacuum, which eliminates the need for an external
source (although the cart can be hooked up to an in-house air source, if one is
available). The air pumps provide pressure from 2 to 75 psi to accommodate a
variety of cell sorting applications. Air pressure is adjusted within BD FACSDiva
software.

Figure 1-2 Fluidics cart

10-L container

5-L container

pressure

gauge

door

access knob

filter

22 BD FACSAria User’s Guide

Containers and Connectors

The fluidics cart holds four 10-L containers (two sheath and two waste), and
three 5-L auxiliary cleaning fluid containers (Figure 1-3). The cart can also
accommodate up to two BD FACSFlow™ 20-L cubitainers in place of the four
10-L containers.

Figure 1-3 Fluidics cart containers

waste port

waste tank

sheath port

sheath tank

To prevent foaming, do not fill the containers with solutions containing a
high concentration of detergent.

The fluidics cart connects directly to the flow cytometer unit via a power cord,
fluid hoses, serial communication cable, and air line (Figure 1-4 on page 24).
Receptacles for the aerosol management and temperature control options are
also located within the connection panel. The position of the fluidics cart is
constrained only by the length of the connecting cables and hoses, which extend
up to 9 feet (2.7 m). Typically, the cart is placed to the left or underneath the
cytometer.

Chapter 1: BD FACSAria Cytometer Components 23

Figure 1-4 Fluidics cart power and fluid line connectors

fluidics cart
power cord

connections

for temperature

control option

AMO

connection

serial communication cable

air input (clear tubing)

sheath input (blue tubing)

aspirated fluid output (yellow tubing)

fluid output (orange tubing)

To connect the fluidics cart to an external air source, open the shutter above the
Communication port, switch on the Auxiliary Air Supply, and attach the external
air line to the air input connector. The external air supply should provide
80–100 psi.

supply
switch

air input
connector

If your laboratory runs the cytometer continuously and does not shut down
at the end of the day, switch on the Auxiliary Air Supply for 15 seconds
every 8 hours (every 4 hours in an extremely humid climate). Doing this
empties the cart condensation trap and prevents excess moisture from
overflowing the trap or causing cart damage.

24 BD FACSAria User’s Guide

Power and Operation

Power to the fluidics cart is supplied by the cytometer—the cart’s fluid pumps are
activated when the cytometer main power switch is turned on (see Power Panel
on page 42).

Power to the fluidics cart is supplied and controlled through the flow
cytometer. The fluidics cart voltage settings have been configured to match
the supply voltage by your service engineer. To properly operate the fluidics
cart, plug the fluidics cart power cord only into the power receptacle on the
cytometer (Figure 1-4 on page 24). Do not plug the power cord directly
into a wall socket. Do not change the input voltage selection jumpers on the
fluidics cart fuse holder.

When the stream is on, air pressure fluctuates between 80–100 psi
(Figure 1-5). A pressure reading of less than 80 psi or greater than 100 psi
indicates that the fluidics cart is not functioning properly. If this occurs,
contact your BD Biosciences service representative for assistance. Do not
operate the cytometer outside the normal air pressure range.

Figure 1-5 Fluidics cart flow gauge

pressure gauge
at approximately
88 psi

See the following sections for more information about the fluidics cart.

• Setting Up the Fluidics Cart on page 112

• Refilling Containers on page 113

• Emptying the Waste on page 114

Chapter 1: BD FACSAria Cytometer Components 25

• Scheduled Maintenance on page 181

• Fluidics Troubleshooting on page 237

Flow Cytometer

The benchtop flow cytometer contains the major components for all three
subsystems (fluidics, optics, and electronics). The BD FACSAria cytometer is
relatively compact, with a much smaller footprint than most sorters with the
same capabilities. The cytometer can be set up on a typical laboratory benchtop
or table, and it requires only a 20-amp electrical outlet. No special facilities are
required.

Figure 1-6 BD FACSAria flow cytometer

flow cell access door

power panel

optics access door

sort collection chamber

sample injection chamber

side door

To view the fluidics components, open the side door and lift the flow cell access
door. To view the optics components, open the optics access door. The power
panel and connectors are found on the left side of the cytometer. Other electronic
components are embedded within the cytometer and do not need user adjustment.

The flow cell access door is equipped with a shutter mechanism that shuts
off the laser light when the door is opened. To ensure there is no interruption
to data acquisition, do not open the door while sorting or recording.

26 BD FACSAria User’s Guide

See the following sections for more information about the flow cytometer.

• Fluidics Components on this page

• Optics System on page 36

• Cytometer Electronics on page 42

Fluidics Components

When the fluidics system is activated, sheath fluid is pumped from the fluidics
cart into a plenum reservoir where the fluidics system is pressurized. From the
reservoir, fluid is pumped into the cuvette flow cell where hydrodynamic focusing
forces particles from the sample injection chamber through the cuvette in a
single-file stream.

Within the cuvette flow cell, laser light is focused on the sample core stream.
Fluorescent molecules excited by the different laser wavelengths are detected by
the optics and analyzed by the electronics. Particles are then either transported to
waste reservoirs via the waste aspirator, or sorted into a receptacle within the sort
collection chamber.

The following fluidics components are described in this section. For more
information about fluidics, see Fluid Movement on page 46.

• Plenum Reservoirs on page 28

• Sample Injection Chamber on page 29

• Cuvette Flow Cell on page 30

• Nozzle on page 31

• Sort Block on page 32

• Sort Collection Chamber on page 35

Chapter 1: BD FACSAria Cytometer Components 27

Plenum Reservoirs

Sheath fluid is pumped from the fluidics cart into a plenum reservoir inside the
side door of the BD FACSAria flow cytometer (Figure 1-7). From there, the fluid
travels to a second reservoir where it is held and pressurized until it flows into the
cuvette flow cell. This system eliminates pressure fluctuations by maintaining
fluid levels and filtering out pump pulsation. As a result, the fluidics pressure
does not vary with the level of fluid in the sheath containers.

Figure 1-7 Plenum reservoirs

Note that the plenum reservoirs can be unscrewed and removed for cleaning, if
needed. See Cleaning the Plenum Reservoirs on page 190.

28 BD FACSAria User’s Guide

Sample Injection Chamber

The sample injection chamber is where sample is introduced into the flow
cytometer. During acquisition, the chamber is pressurized to force sample toward
the cuvette flow cell.

Samples can be agitated and temperature-controlled within the sample injection
chamber using controls in the software (see Fluidics Controls on page 81). You
can view the amount of fluid remaining in your sample tube by pressing the
chamber light button shown in Figure 1-8.

Do not use the chamber light for long periods with samples stained with
light-sensitive reagents.

Figure 1-8 Sample injection chamber and tube holders

sample
injection
chamber

chamber
light

15 mL

12 x 75 mm

tube holder

loading port

1 mL

microtube

tube holders

A variety of tube holders are provided with the cytometer to accommodate tubes
from 15-mL centrifuge tubes to 1.0-mL microtubes (Figure 1-8). (For a list of
compatible tubes, see Labware on page 248.) To load a tube, install the
appropriate-size tube holder in the loading port, and place a tube in the holder.
When the Load button is clicked in the software (see Acquisition Controls on
page 91), the loading port rises to enclose the tube within the chamber.

After a tube is loaded, the Load button changes to Unload. Click the Unload
button to lower the loading port after data has been recorded. After each tube is
unloaded, sheath fluid flushes the sample tubing inside and out to reduce

Chapter 1: BD FACSAria Cytometer Components 29

potential sample carryover. Using a special cleaning mode, the entire sample
injection chamber can be decontaminated with ethanol. See Clean Bulk Injection
Chamber on page 191 for more information.

To prevent injury from moving parts, keep your hands and clothing away
from the loading port when a tube is loading or unloading. Do not place
objects under the loading port.

Cuvette Flow Cell

The cuvette flow cell is the heart of the BD FACSAria cytometer (Figure 1-9).
Within the flow cell, hydrodynamic focusing forces particles through the cuvette
in a single-file stream, where laser light intercepts the stream at the sample
interrogation point.

Figure 1-9 Cuvette flow cell

flow cell

interrogation point

nozzle

laser light intercepting flow cell

The unique flow cell design permits particles to flow through the cuvette at a low
velocity (approximately 13 cm/sec for the high Sort Setup), allowing longer
exposure to laser energy. The cuvette is gel-coupled to the fluorescent objective
lens to transmit the greatest amount of emitted light from the interrogation point
to the collection optics (see Optics System on page 36). After passing through the
cuvette, the stream is accelerated (to approximately 69 cm/sec with the 70-µm
nozzle) as it enters the nozzle tip, where the drop drive breaks the stream into
droplets for sorting.

30 BD FACSAria User’s Guide

Nozzle

The BD FACSAria cytometer is provided with two adjustable nozzles, 70 and
100 μm, that accommodate a variety of particle sizes. The nozzle is keyed to a
fixed position at the end of the cuvette. Because the nozzle is below the
interrogation point, optical alignment is not affected when the nozzle is changed.

Figure 1-10 Nozzle

O-ring

Chapter 1: BD FACSAria Cytometer Components 31

Sort Block

After leaving the nozzle, particles pass through the sort block where they are
either transported to waste via the waste aspirator, or sorted into a collection
device in the sort collection chamber. The sort block houses the high-voltage
deflection plates, along with the aspirator and aspirator drawer (Figure 1-11).

Figure 1-11 Sort block with door open

deflection plate

adjustment screws

aspirator

aspirator drawer

sort collection

device

Note that the entire sort block assembly can be rotated on a fixed pivot point to
adjust the position of the stream in the waste aspirator. If the keyed stream
position differs between an old nozzle and a new one, the stream might not hit
the center of the aspirator after the nozzle is changed. In this case, you can
change the angle of the sort block by loosening the adjustment screws on either
side of the deflection plates and rotating the sort block. Tighten the screws when
the stream is re-centered in the aspirator.

32 BD FACSAria User’s Guide

Deflection Plates

The high-voltage deflection plates are used to deflect side streams during sorting.
The plates are turned on and off using the Voltage control in the Side Stream
window (see Side Stream Formation on page 67). A red warning light is
illuminated whenever the plate voltage is on (Figure 1-13 on page 34).

A 12,000-volt potential exists between the deflection plates when they are
on. Contact with the charged plates results in serious electrical shock. Do
not touch the deflection plates when the voltage warning light is
illuminated, or when the software indicates that the plate voltage is on. The
plates remain energized even when the sort block door is opened.

Aspirator Drawer

The aspirator drawer keeps the sort collection tubes covered until sorting begins
(Figure 1-12). You can open and close the drawer using a control in the Sort
Layout or Side Stream window (see Using Sorting Controls on page 100). When
the Sweet Spot is on and a clog is detected during sorting, the drawer closes
automatically to protect your sort collection tubes.

To avoid pinching your hands or fingers in the drawer, keep your hands
away from the sort block during sorting.

Figure 1-12 Aspirator drawer closed (left) vs open (right)

Chapter 1: BD FACSAria Cytometer Components 33

Aerosol Management

During sample acquisition and sorting, the sort block door should be kept closed
to help contain potential aerosols (Figure 1-13).

Cell sorters that use droplet generation methods, like the BD FACSAria,
can produce aerosols around the sample stream. Inhalation or contact with
aerosols exposes you to biologically transmissible diseases. When acquiring
biohazardous samples, follow universal precautions at all times. Keep the
sort block door closed during sorting. If you need to access the sort block,
turn off the stream before opening the door.

Figure 1-13 Sort block with door closed

deflection plates
warning light

sort block

sort collection
chamber

Additional aerosol removal is provided by the BD™ Aerosol Management
Option (AMO). The AMO is an optional device that evacuates the sort collection
chamber during sorting. The AMO is equipped with a 0.01-µm pore, ultra-low
penetrating air (ULPA) filter to trap aerosolized particles. The AMO pump
evacuates the sort collection chamber 16 times per minute when operating at
20% capacity in normal evacuation mode, and 69 times per minute at 100%
capacity in rapid evacuation mode.

34 BD FACSAria User’s Guide

Sort Collection Chamber

Collection devices are installed in the sort collection chamber to collect sorted
samples during sorting. The following collection devices are provided with the
cytometer to accommodate a variety of tube sizes:

• Four-way 1-mL collection tube holder

• Four-way 12 x 75-mm collection tube holder

• Two-way 12 x 75-mm collection tube holder

• Two-way 15-mL collection tube holder

An automated cell deposition unit (ACDU) that sorts into multiwell plates and
onto microscope slides is available as an option. BD Biosciences also offers a
temperature-control option to maintain the temperature of sorted samples during
sorting.

Figure 1-14 Sort collection devices

four-way

12 x 75 mm

two-way 15 mL

four-way 1 mL

plate loadercollection devices

The sort collection chamber door should be kept closed when sorting into a plate.
The door keeps the chamber free of dust and other air-borne particles, and seals
the chamber during aerosol evacuation for cytometers equipped with the AMO.

Chapter 1: BD FACSAria Cytometer Components 35

Optics System

The BD FACSAria cytometer uses innovative designs for both the excitation
optics and collection optics. The optics can be viewed by opening the optics
access door and the flow cell access door. See the following sections for more
information.

The flow cell access door is equipped with a shutter mechanism that shuts
off the laser light when the door is opened. To ensure there is no
interruption to data acquisition, do not open the door while sorting or
recording.

Excitation Optics

The excitation optics consist of lasers, fiber optic cables, beam-shaping prisms,
and an achromatic focusing lens, as shown in Figure 1-15 on page 37. For
information about how signals are generated, see Signal Generation on page 50.

The BD FACSAria cytometer uses low-powered, air-cooled and solid-state lasers
that do not have special power and cooling requirements. The cytometer comes
standard with 488-nm and 633-nm lasers. A third 405-nm laser is available as an
option.

a

Laser

Coherent® Sapphire™
Solid State

JDS Uniphase™ HeNe
Air Cooled

Point Source Violet
Solid State (optional)

a. Measured out of fiber optic cable
b. Patents: PerCP—US 4,876,190; APC-Cy7—US 5,714,386

Wavelength

(nm)

488 (blue) 13 FITC, PEb, PE-Texas Red®,

633 (red) 11 APC, APC-Cy7

405 (violet) 30 Alexa Fluor®, Cascade Blue®,

Min. Power

(mW)

Commonly Used Fluorochromes

or Dyes

PerCP, PerCP-Cy

PE-Cy7, PI

Pacific Blue™, DAPI, Hoechst

™5.5,

36 BD FACSAria User’s Guide

Fiber optics direct the laser light in a precise and constant manner onto beam­shaping prisms, which in turn transmit the laser light to a focusing lens. The lens
focuses the laser light onto the sample core stream within the cuvette flow cell
(Figure 1-15). The lasers are positioned on the sample stream for optimal
generation of signals. Since the optical pathway and sample core stream are
fixed, optimization is constant from day to day.

Lasers emit intense, coherent electromagnetic radiation that can cause
irreparable damage to human skin and eyes. To prevent retinal burns and
possible blindness, do not remove laser shielding, adjust laser controls, or
attempt to service the cytometer any place where laser warning labels are
attached. See Precaution Labels on page xviii for the placement of laser
warning labels.

Figure 1-15 Excitation optics pathway (behind laser shielding)

prisms

fiber optics

prisms

focusing lens

cuvette
flow cell

upper
camera

Chapter 1: BD FACSAria Cytometer Components 37

Collection Optics

From the cuvette flow cell, laser light is collected by a fluorescent objective lens
that is gel-coupled to the cuvette to transmit the maximum amount of light. The
lens collects and focuses fluorescent light emitted at each of the laser focal points
onto individual collection fibers. These fibers transfer the emitted light to the
collection optics, as shown in the following figure.

individual
collection

fibers

cuvette

fluorescent objective lens

stream

The collection optics are set up in octagon- and trigon-shaped arrays that are
engineered to maximize signal detection from each laser. This is accomplished by
transmitting the highest wavelengths to the first photomultiplier tube (PMT), and
reflecting lower wavelengths to the next PMT through a series of longpass
dichroic mirrors. Bandpass filters in front of each PMT allow fine-tuning of the
spectral wavelengths that need to be collected. Since reflection is more efficient
than transmittance, this design greatly increases the multicolor detection
capabilities of the cytometer.

38 BD FACSAria User’s Guide

Detectors

A standard system is equipped with an octagon containing six PMTs, which
detect light from the 488-nm (blue) laser, and a trigon containing two PMTs,
which detect light from the 633-nm (red) laser. The third-laser option adds an
additional trigon containing two PMTs to detect light from the 405-nm (violet)
laser. Optical upgrades are available to fill the remaining PMT slots for a two- or
three-laser system. A fully-equipped system can detect up to 13 fluorescent
parameters and two scatter parameters at a time.

Figure 1-16 Fully loaded detector arrays

trigons

violet-laser

signal

blue-laser

signal

red-laser

octagon

signal

At installation, the octagon and trigon arrays are set up with the filter and mirror
combinations shown in Table 1-1 on page 40. You can rearrange these
configurations according to the type of fluorochromes in your experiment. See
Application Options on page 250 for more information.

Chapter 1: BD FACSAria Cytometer Components 39

Table 1-1 Default setup for detector arrays

Detector Array

(Laser)

Octagon

PMT LP Mirror BP Filter Intended Fluorochrome or Dye

A 735 780/60 PE-Cy7
(488-nm blue
laser)

B 655 695/40

675/20

C 610 616/23 PE-Texas Red®

D 556 585/42 PE or PI

E 502 530/30 FITC

F — 488/10

Trigon
(633-nm red
laser)

Trigon
(405-nm violet
laser)

a. The optical holder for the 488/10 BP filter also includes a 1.0 ND filter.

A 735 780/60 APC-Cy7

B — 660/20 APC

A 502 530/30 Alexa Fluor® 430

B — 450/40 Cascade Blue®, Pacific Blue

a

PerCP-Cy5.5 or PI
PerCP

Side scatter (SSC)

™,

DAPI, Hoechst,
Alexa Fluor® 405

40 BD FACSAria User’s Guide

Stream-Viewing Optics

The BD FACSAria cytometer is equipped with additional optical components
that are used to view the stream (Figure 1-17).

• The upper camera generates an image used to monitor drop formation. It is
focused on the stream, just below the nozzle, to provide an image of the
drop breakoff.

• The lower camera generates an image used for the BD FACS™ Accudrop

*

option. It enhances the ability to see side streams and assists in setting an
accurate drop delay value.

Figure 1-17 Stream-viewing optics

upper

camera

diode laser

micrometer dial

lower camera
viewing window

Special image-processing software allows you to view the stream images from
each camera within separate windows in BD FACSDiva software. See Sorting on
page 62 for more information about viewing the streams and to learn how
Accudrop components are used to determine the drop delay.

* US Patent No. 6,372,506

Chapter 1: BD FACSAria Cytometer Components 41

Cytometer Electronics

The electronic components consist of power controls and connectors along with
processing boards in the card cage. This section describes only user-adjustable
cytometer electronics. For more information about the electronics, see Signal
Detection on page 52.

Power Panel

Power to the cytometer, lasers, and fluidics cart is supplied by a power cord
plugged directly into a standard electrical outlet. There are no special facilities
requirements. The main power button turns on the cytometer and fluidics cart,
and enables the laser power buttons. To turn on a laser, press the corresponding
button. The button lights up when the power is on.

Do not turn on the lasers until you have activated the stream. Running the
lasers without activating the stream can degrade the performance of the
cuvette flow cell.

Figure 1-18 Flow cytometer power panel

main power

power cord circuit breaker

The cytometer circuit breaker is located next to the power cord (Figure 1-18).
The switch will need to be reset if there is a power surge in the laboratory.

42 BD FACSAria User’s Guide

laser power

Emergency Stop Button

The emergency stop button to the right of the loading port (Figure 1-19) is a
safety feature that can be used to halt the movement of the loading port and
ACDU stage. The button lights up when a tube is loading to remind you to keep
your hands away from the loading port.

Figure 1-19 Emergency stop button

emergency stop button

The following occur when this button is pushed:

• The tube is unloaded from the sample injection chamber.

• The ACDU stage (if in use) stops moving.

• The stream is turned off.

• The deflection plate voltage is turned off.

• The aspirator drawer (if open) closes to protect sort collection tubes.

• A warning message appears on the screen.

Do not reset the button until the message appears. To do so, turn the button
clockwise until the light turns off and the button returns to its original position.

NOTE The emergency stop button does not turn off the lasers or shut down the
cytometer main power.

!

Chapter 1: BD FACSAria Cytometer Components 43

Workstation

Data acquisition and analysis, as well as most BD FACSAria cytometer functions,
are controlled by BD FACSDiva software on a third-party PC workstation. The
workstation includes a desktop computer, one or two monitors, and a color
printer, and is equipped with the following applications:

• Microsoft Windows operating system

• BD FACSDiva software, version 6.1 or later

- Data acquisition and analysis

- Automatic fluidics startup, shutdown, and cleaning modes

- Sort setup, stream monitoring, clog detection, and recovery

• Supporting documentation for the software

For information about BD FACSAria–specific software features, see Chapter 2,
Theory of Operation, and Chapter 3, Using BD FACSDiva Software. For general
software information, refer to the BD FACSDiva Software Reference Manual.

 Tip For easy access to the BD FACSDiva Software Reference Manual, select

Help > Literature > Reference Manual.

44 BD FACSAria User’s Guide

Theory of Operation

This chapter describes how the BD FACSAria cytometer works and how
BD FACSDiva software components are used to operate different system
components. For a general overview of the software, see Chapter 3.

See the following sections for a description of these BD FACSAria functions:

• Fluid Movement on page 46

• Signal Generation on page 50

• Signal Detection on page 52

• Electronic Processing on page 59

• Sorting on page 62

2

45

Fluid Movement

The fluidics system is responsible for moving particles from the sample injection
chamber through the cuvette flow cell for interrogation, and then to waste or into
a collection device during sorting (Figure 2-1). The following sections describe
the controls used to move fluids through the BD FACSAria fluidics system.

Figure 2-1 Fluidic movement via the fluidics system

Sheath Flow

sheath tank

sheath filter

plenum reservoirs HPLC valve

cuvette flow

cell

interrogation point

waste aspirator

Sample Flow

sample tube

sample injection

chamber

sample collection

chamber

collection tube

or plate

Sheath Flow

When you turn on the cytometer, the fluidic pumps start up. The fluidics system
is activated when you choose the Fluidics Startup command in BD FACSDiva
software. During fluidics startup, sheath fluid is pumped from the sheath
container through a 0.22-μm filter into the plenum reservoirs. The reservoirs are
pressurized to deliver sheath fluid to the cuvette flow cell at a constant pressure.
You can view the current sheath pressure using the Cytometer > Sheath Pressure
command (Figure 2-2 on page 47).

46 BD FACSAria User’s Guide

Figure 2-2 Sheath pressure level

After fluidics startup, sheath flow is controlled using the Stream button in the
Breakoff window, which is labelled with the chosen sort pressure (Figure 2-3).
When clicked, the button changes from a red “X” to a green checkmark, and
sheath flows through the cuvette flow cell at the rate that is specified in the
Sheath Pressure window. (See Drop Formation on page 63 for a complete
description of the Breakoff window.)

Figure 2-3 Stream control in Breakoff window

As a general rule, the sheath pressure level is set by choosing a Sort Setup option
from the Sort menu, rather than by adjusting the Sheath Pressure control. Each
Sort Setup option is optimized at a preset sheath pressure. If you change the
sheath pressure, a multitude of other values will be affected, including the drop
drive frequency, drop charge levels, laser delay, area scaling factor, and other
values. For more information, see Sort Setup on page 94.

Chapter 2: Theory of Operation 47

Sample Flow

Sample is introduced into the cuvette when the Load button is clicked in the
Acquisition Dashboard (Figure 2-4). After Load is clicked, the loading port rises
to enclose the tube within the sample injection chamber. The chamber is
automatically pressurized and the chamber pressure forces sample through the
sample line into the cuvette flow cell. To stop sample flow after a tube is loaded,
click the Stop Acquiring button.

The sample flow rate is specified using the Flow Rate field in the Acquisition
Dashboard. You can adjust the Flow Rate from 1–11, which corresponds to
approximately 10–120 μL/min.

Figure 2-4 Load button and Flow Rate field in Acquisition Dashboard

Note that the relatively longer sample tubing on the BD FACSAria cytometer
results in a different flow rate between cells and beads. Thus, absolute counting
using BD Trucount™ beads can give erroneous results.

48 BD FACSAria User’s Guide

Hydrodynamic Focusing

In the flow cell, pressurized sheath fluid surrounds the sample fluid to
hydrodynamically focus the core stream of suspended cells into the center of the
cuvette, where the particles are intercepted by the laser beam.

The difference in pressure between the sheath fluid and the sample fluid can be
used to vary the diameter of the sample core. A lower difference results in a relatively
narrow core stream, while a higher difference results in a wider sample stream.

sheath flow

sample flow

sheath flow

laser beams

low flow rate high flow rate

sheath flow

sample flow

sheath flow

laser beams

Ideally, you want the core stream at its minimum diameter so that cells pass
through the laser beam in a single-file stream. However, depending on your
application, a lower resolution might be acceptable in order to acquire the data
more quickly. For example, a higher flow rate is generally used for qualitative
measurements such as immunophenotyping—the data is less resolved but is
acquired more quickly. A lower flow rate is generally used in applications where
greater resolution is critical.

Chapter 2: Theory of Operation 49

Signal Generation

The following sections describe how signals are generated when cells or particles
intercept the laser within the cuvette flow cell.

Light Scatter

When a cell or particle passes through a focused laser beam, laser light is
scattered in all directions. Light that scatters axial to the laser beam is called
forward scatter (FSC). Light that scatters perpendicular to the laser beam is
called side scatter (SSC). FSC and SSC are related to certain physical properties of
cells:

• FSC—indicates relative differences in the size of the cells or particles

• SSC—indicates relative differences in the internal complexity or granularity
of the cells or particles

side scatter

forward scatter

light source

50 BD FACSAria User’s Guide

Fluorescent Signals

When cells or particles stained with fluorochrome-conjugated antibodies or other
dyes pass through a laser beam, the dyes can absorb photons (energy) and be
promoted to an excited electronic state. In returning to their ground state, the
dyes release energy, most of which is emitted as light. This light emission is
known as fluorescence.

Fluorescence is always a longer wavelength (lower-energy photon) than the
excitation wavelength. The difference between the excitation wavelength and the
emission wavelength is known as the Stokes shift. Some fluorescent compounds
such as PerCP exhibit a large Stokes shift, absorbing blue light (488 nm) and
emitting red light (675 nm), while other fluorochromes such as FITC have a
smaller Stokes shift, absorbing blue light and emitting green light (530 nm).

The emission spectra for some commonly used fluorochromes are shown in
Figure 2-5. See Table 1-1 on page 40 for the corresponding detectors.

Figure 2-5 Emission spectra of commonly used fluorochromes

100%

Normalized Intensity

0%

Cascade

400

Blue

Hoechst

Pacific

Blue

FITC

500

Alexa

Fluor 430

PI

RPE

600 700 800

Wavelength (nm)

APC PerCP PerCP-Cy5.5

Chapter 2: Theory of Operation 51

PE-Cy7

Signal Detection

From the cuvette flow cell, scattered and fluorescent light is collected by the
fluorescent objective lens. The lens collects and focuses fluorescent light emitted
at each of the laser focal points onto individual collection fibers. These fibers
transfer the emitted light to the individual detector arrays.

The following sections describes how laser light is detected and translated into
signals that can be displayed in a plot.

Detector Arrays

There are two types of detector arrays in the BD FACSAria flow cytometer: an
octagon and trigons. The octagon detects SSC and up to seven fluorescent signals
excited by the 488-nm (blue) laser. The trigons detect fluorescent signals excited
by the 633-nm (red) and 405-nm (violet) lasers, respectively. Each trigon can
detect up to three fluorescent channels. (An optical upgrade is required to fill all
detection channels.)

Figure 2-6 Octagon and trigon detector arrays

blue-laser signalred-laser signalviolet-laser signal

Each detector array houses dichroic and bandpass filters, which steer and filter
the emitted light, and photomultiplier tubes (PMTs), which detect light signals.

52 BD FACSAria User’s Guide

Filters

Optical filters modify the spectral distribution of light scatter and fluorescence
directed to the detectors. Three kinds of filters are used in the detector arrays:
longpass (LP) filters are used to steer light between the detectors within a detector
array, while bandpass (BP) and neutral density (ND) filters allow fine-tuning of
the spectral wavelengths that need to be collected (Figure 2-7).

Figure 2-7 Detectors and filters in octagon array

longpass

bandpass

PMT

Chapter 2: Theory of Operation 53

Longpass Filters

Longpass (LP) filters pass wavelengths longer than the filter rating and reflect
shorter wavelengths. For example, a 500 LP filter permits wavelengths longer than
500 nm to pass through it and reflects wavelengths shorter than 500 nm
(Figure 2-8).

Figure 2-8 Light reflection by longpass filter

longpass

% transmission

wavelength (nm)

Dichroic filters that are used to direct different color light signals to different
detectors are called dichroic mirrors or beam splitters.

Although dichroic mirrors have the properties of LP optical filters, you cannot
necessarily use any type of LP filter as a beam splitter. A beam splitter must have
a surface coating that reflects certain wavelengths, but many types of LP filters
are absorbance filters that do not have any specific reflective characteristics. Also,
optical filters and beam splitters are rated at a specific angle of incidence. When
used as a beam splitter, they are placed at an angle relative to the light source.
Their optical properties are therefore designed for that angle of incidence.

The detector arrays use LP dichroic mirrors to steer progressively shorter
wavelengths of light to the next PMT in the array. For example, in the octagon
array, light first passes through a 735 LP filter in the A position, followed by a
655 LP filter in the B position. Thus, wavelengths longer than 735 nm are
detected at PMT-A; all wavelengths <735 nm are reflected to PMT-B. Wavelengths
between 655 nm and 735 nm are detected at PMT-B; all wavelengths <655 nm are
reflected to PMT-C, and so on. For a list of the longpass filters used in the
detector arrays, see Table 1-1 on page 40.

54 BD FACSAria User’s Guide

Bandpass Filters

Bandpass (BP) filters transmit a relatively narrow range or band of light.
Bandpass filters are typically designated by two numbers. The first number
indicates the center wavelength and the second refers to the width of the band of
light that is passed. For example, a 500/50 BP filter transmits light that is
centered at 500 nm and has a total bandwidth of 50 nm. Therefore, this filter
transmits light between 475 and 525 nm (Figure 2-9).

Figure 2-9 Light transmittance by bandpass filters

bandpass

% transmission

wavelength (nm)

Discriminating (DF) filters have the same general function—they transmit a
relatively narrow band of light. The principal difference between BP and DF
filters is their construction. DF filters have more cavities or layers of optical
coatings, resulting in a steeper transmission curve than the curve for a BP filter.
This steep slope means that a DF filter is better at blocking light outside the rated
bandwidth of the filter. See Figure 2-10 on page 56.

Chapter 2: Theory of Operation 55

Figure 2-10 Bandpass (BP) vs discriminating (DF) filters

BP 500/50 filter

DF 500/50 filter

% transmission

wavelength (nm)

In the detector arrays, DF filters block high-intensity laser light and filter the
remaining light to ensure that only the required wavelengths reach their intended
detector. For example, in the octagon array, PMT-A has a 780/60 DF filter in
front of it, which transmits light of 750–810 nm. Thus, the only wavelengths that
will reach the A detector are those between 750 and 810 nm.

For optimal detection of fluorescent light, a bandpass filter must always be
installed in front of each detector. For a list of the bandpass filters used in the
detector arrays, see Table 1-1 on page 40.

56 BD FACSAria User’s Guide

Neutral Density Filters

Neutral density (ND) filters transmit a fixed percentage of light, reducing the
transmitted intensity of all wavelengths equally. ND filters are neutral with
respect to wavelength.

100%

ND1

10%

The ND filters on the BD FACSAria allow approximately 10% of the light to be
transmitted. You can find them in front of the FSC and SSC detectors.

For applications involving small particles (eg, bacteria or platelets), you might
need to remove the FSC ND filter. See Removing/Installing the FSC ND Filter on
page 254. For applications involving large particles where events appear off scale
on the FSC axis with a gain of zero, the FSC ND filter is needed to decrease the
FSC signal and keep the events on scale.

Chapter 2: Theory of Operation 57

Detectors

Detectors within each detector array convert light signals into electrical signals
that can be processed by the electronics system.

There are two types of signal detectors in the BD FACSAria flow cytometer: the
photodiode and photomultiplier tubes (PMTs). The photodiode is less sensitive to
light signals than the PMTs, thus is used to detect the stronger FSC signal. The
photodiode detects FSC light from the blue laser, and is stored outside the
detector arrays. PMTs are used to detect the weaker signals generated by SSC and
all fluorescence channels. The octagon can hold up to eight PMTs; each trigon
can contain up to three PMTs.

Each PMT detects only one fluorochrome at a time. In BD FACSDiva software,
the Cytometer Configuration window lets you define which fluorochromes or
cell parameters will be measured at each PMT detector. If more than one
fluorochrome is measured using the same PMT, you can add additional
parameters to your configuration and choose the appropriate fluorochrome
within your software experiment. See Cytometer Configuration on page 86 for
more information.

Figure 2-11 Example BD FACSAria cytometer configuration

58 BD FACSAria User’s Guide

Electronic Processing

As cells or other particles pass through the focused laser beams, they scatter the
laser light and can emit fluorescence. Because each laser beam is focused on a
small spot and particles move rapidly through the flow cell, the scatter or
fluorescence emission has a very brief duration—only a few microseconds. The
PMTs convert this brief flash of light into an electrical signal called a pulse
(Figure 2-12).

Figure 2-12 Anatomy of a pulse

1

2

3

A pulse begins when a particle enters the laser beam. At this point, both the

1

beam intensity and signal intensity are low.

The pulse reaches a maximum intensity or height when the particle reaches the

2

middle of the beam, where the beam and signal intensity are the brightest. The
peak intensity, or height of the pulse, is measured at this point.

As the particle leaves the beam, the pulse trails off.

3

Chapter 2: Theory of Operation 59

Pulse Parameters

A parameter is a pulse property that is
generated by a single photomultiplier tube or
photodiode, measuring fluorescent or
scattered light. You can measure three
characteristics of a pulse: the area, height, or
width. The pulse height measures the
maximum digitized value for the pulse; the
pulse area calculates the sum of all height
areas for the pulse; and the pulse width
calculates .

area

--------------

height

64000×

You can select which pulse characteristics you
want to measure for each parameter using the
Parameters tab. Pulse area (A) is measured by default. The Parameters tab also
contains voltage controls that allow you to amplify signals by applying a voltage
to PMTs or an electronic gain to the FSC signal. As the voltage is increased, the
detector sensitivity increases, resulting in increased signal. As the voltage is
decreased, the detector sensitivity decreases, resulting in decreased signal.

signal

intensity

pulse

area

pulse
width

pulse height

time

Digital data is displayed on an 18-bit linear scale, from 2.6–262,144. Select the
Log checkbox to convert the display to a log scale. The Experiment Inspector
contains an option to display log data on a four- or five-decade scale. (Refer to
the BD FACSDiva Software Reference Manual for more information.)

60 BD FACSAria User’s Guide

Laser Delay

Sample interrogation takes place within the cuvette flow cell. As described in
Optics System on page 36, fiber optic cables are used to direct laser light through
a series of prisms that focus each laser on the stream at a separate position. This
allows optimal detection of fluorescent signals from each laser with minimal
cross-contamination from the other beams.

In a three-laser system, the red laser intercepts the stream first, followed by the
blue and then the violet laser. Because the laser signals are spatially separated,
there is a slight delay between the detection of each laser’s signal (Figure 2-13).

Figure 2-13 Signal separation over time

cell enters and leaves
the first laser intercept

cell enters and leaves the
second laser intercept

cell enters and leaves
the third laser intercept

time

red

blue

violet

The delay factor in BD FACSDiva software is used to realign the signals so they
can be measured and displayed on the same time scale. Note that signals are
aligned with respect to the blue laser, so the red laser signals always have a
negative delay value.

Chapter 2: Theory of Operation 61

Sorting

During sorting, drop drive energy is applied to the stream to break it into highly
uniform droplets. Droplets detach from the stream a few millimeters downstream
from the nozzle. The time between when a particle intercepts the laser and when
it reaches the droplet breakoff point is determined using BD FACS Accudrop
technology (see Drop Delay Overview on page 69).

When a particle is detected and meets the predefined sorting criteria, an electrical
charge is applied to the stream just as the droplet containing that particle breaks
off from the stream. Once broken off from the stream, the droplet—now
surrounded by air—still retains its charge. The charged droplet passes by two
strongly charged deflection plates. Electrostatic attraction and repulsion cause
each charged droplet to be deflected to the left or right, depending on the
droplet’s charge polarity. Uncharged droplets are not affected by the electric field
and pass down the center to the waste aspirator. See Figure 2-14 on page 63.

The following sections describe how the BD FACSAria cytometer creates and
charges drops, how the drops are deflected, and how sorting populations are
identified.

62 BD FACSAria User’s Guide

Figure 2-14 Sorting

1

2

3

4

5

6

Charge is applied via the stream-charging wire in

1

the barb.

Sample generates light scatter and fluorescent

2

signal; signal is analyzed.

The charged droplet breaks off.

3

Deflection plates attract or repel the charged

4

droplet.

Uncharged droplets pass to waste.

5

Charged drops containing particles of interest are

6

collected.

Drop Formation

The BD FACSAria cytometer is unique in that drop drive energy is constantly
applied to the stream; droplets form as soon as you turn on the stream. Sample
interrogation takes place upstream of the stream vibration so that analysis is not
affected by the drop drive.

A drop breakoff image is created using an LED strobe and a video camera. The
image is displayed in the Breakoff window. (See Breakoff Window on page 64.)
Patented Sweet Spot technology
feedback to the appropriate cytometer controls.

* US Patent No. 5,700,692

*

analyzes the drop breakoff image and provides

Chapter 2: Theory of Operation 63

Breakoff Window

The upper camera transmits an image of the drop breakoff to the
Breakoff window, where video image processing software
converts the visual characteristics of the image into numerical
properties. The drop breakoff is analyzed for two key features:
Drop 1 and Gap.

Drop 1 is defined as the number of pixels from the top of the
image to the center of gravity of the first broken-off drop. A thin
gray line on the image is used to identify this drop. The number
shown to the right of the Drop 1 field at the bottom of the
window is the actual pixel location of the gray line. The number
entered into the Drop 1 field is the user-defined Drop 1 target.

The Gap is defined as the number of pixels from the first
discontinuity in the stream to the next stream object, generally the
first broken-off drop. A gray line of varying thickness represents
the Gap. The number shown to the right of the Gap field is the
pixel width of the gray line. The number entered into the Gap

Drop 1

field is a user-defined target.

When the Sweet Spot is turned on, the drop drive amplitude
(Ampl.) is automatically adjusted to approximately match the
target Drop 1 and Gap values. The amplitude is initially adjusted
in larger increments until Drop 1 is achieved. The amplitude is
then adjusted in smaller increments until the cytometer attains the
target Gap. The Sweet Spot feature will make adjustments as
necessary to maintain the required breakoff conditions
throughout the day.

The Sweet Spot performs two other functions during sorting.
When sorting, if the Drop 1 or Gap are out of range, sorting is
paused until the values are back within range. This ensures that
sorting occurs only under the proper breakoff conditions. If a
more severe problem such as a clog is detected by the Sweet Spot,
the stream is shut off and sorting is stopped: the deflection plates
shut off, the aspirator drawer closes, and the sample tube is unloaded.

target

value

Gap

actual

value

64 BD FACSAria User’s Guide

Pre-programmed values can be downloaded to the Breakoff window by selecting
one of the nozzle sizes (70, 85, 100, 130 micron) from the Sort > Sort Setup menu.

Note that changes to values in the Sort Setup windows (Breakoff and Side
Stream) are automatically saved. At startup, the last settings used on the
cytometer are restored, except the Stream and Sweet Spot controls which always
default to off. Default values can be restored by selecting Sort > Sort Setup >
Restore Defaults. For more information, see Sort Setup on page 94.

Table 2-1 Breakoff window controls

Control Description

Stream button Turns the stream on and off .

Sweet Spot
button

on off

Amplitude field Adjusts the amplitude or intensity of the drop drive,

Frequency field Determines the number of drops formed per second and

Enables automatic adjustment of the drop drive
amplitude to maintain the stability of the breakoff
point.

When the Sweet Spot is on, the Amplitude and
Frequency fields are disabled; the amplitude is
automatically adjusted by the software. To enable the
fields, turn off the Sweet Spot.

from 1.0–80.0 volts.

The drop drive amplitude determines the breakoff point.
A higher amplitude value results in a shorter stream
breakoff; a lower amplitude results in a longer stream
breakoff. Typically, the amplitude is set once, at the
beginning of a sorting experiment, and then maintained
via the Sweet Spot.

the size of the drops. (Drop size is also influenced by
the nozzle size.)

The drop drive frequency can be adjusted from 1.0–

102.0 kHz. The higher the frequency, the more drops
are generated per second and the smaller the drops. The
lower the frequency, the fewer drops generated per
second and the larger the drops.

Chapter 2: Theory of Operation 65

Table 2-1 Breakoff window controls (continued)

Control Description

Frequency field
(continued)

Drop 1 field The distance between the top of the image and the center of the first

Gap field The gap between the stream breakoff and the top of the first drop.

In general, the drop drive frequency should not need adjustment.
BD Biosciences recommends using the default values that are entered
with each Sort Setup mode.

broken-off drop, from 100–600 pixels.

When you input a value and turn on the Sweet Spot, the cytometer
automatically adjusts the amplitude to attain your target value.

Note that not all Drop 1 targets are attainable. The Drop 1 value
jumps in whole increments of approximately 57 pixels according to
your drop spacing.

The same Drop 1 setting should be used from day to day. A difference
of up to 10 pixels between the target value and the actual value is
acceptable.

The default Gap setting is 6 pixels, and in general, it should never be
adjusted. Differences of 1–2 pixels between the target value and the
cytometer-generated value are acceptable.

Typically, when setting up for sorting, you use the Amplitude to set the required
drop breakoff, and copy the generated Drop 1 value to the target field. Then, you
turn on the Sweet Spot to maintain the drop breakoff. When the Sweet Spot
detects a >1-pixel difference between the target Gap setting and the actual Gap, it
adjusts the Amplitude to reduce the Gap. When a >2-pixel difference is detected,
the Sweet Spot stops sorting temporarily (stops charging the stream) until the
Gap is restored. Once the Gap is back within range, sorting automatically
resumes.

For information on setting the breakoff, see Setting Up the Breakoff on page 110.

66 BD FACSAria User’s Guide

Side Stream Formation

Side streams are formed when the voltage is on and you are sorting, or when you
click Voltage, then Test Sort in the Side Stream window.

The Side Stream window displays an image of the side streams as transmitted by
the lower camera. In addition to the stream image, the Side Stream window
contains the controls used to adjust electrical charges and to determine the drop
delay using Accudrop, as described in Drop Delay Overview on page 69.

Figure 2-15 Side Stream window

Controls in the Side Stream window are described in Table 2-2 on page 68. You
can send different values to the Side Stream window by choosing a setting from
the Sort > Sort Setup menu.

Note that changes to values in the Sort Setup windows (Side Stream and
Breakoff) are automatically saved. At startup, the last settings used on the
cytometer are restored, except the states of the Voltage, Test Sort, and Optical
Filter buttons, which always default to off. Default values can be restored by
selecting Sort > Sort Setup > Restore Defaults. For more information, see Sort
Setup on page 94.

Chapter 2: Theory of Operation 67

Table 2-2 Side Stream window controls

Control Description

Voltage button Turns the plate voltage on and off.

off

on

Test Sort button Generates test side streams based on test sort pulses.

on off

Optical Filter button Controls the position of the optical filter in front of the lower

(Accudrop) camera.

in out

Attenuation button Decreases the amplitude of the drop drive. At startup,

attenuation defaults to off. As a general rule, turn on
attenuation when sorting below 30 psi.

off on

Waste Drawer button Moves the aspirator drawer in or out depending on its current

state. The default state is in. For more information, see
Aspirator Drawer on page 33.

closed open

Drop Delay field Sets the amount of time between when an event is measured

and the breakoff point, from 10–140 drops. The drop delay
value determines which drop will be deflected.

The drop delay value is set experimentally using BD FACS
Accudrop technology.

Auto Delay Opens the Auto Drop delay dialog.

Voltage sliders (far
left, left, right, far

Set the percentage of charge to be applied to the corresponding
stream (as a percentage of maximum).

right)

Voltage Center slider Adjusts the relative voltage between the left and right plates

which moves the streams slightly to the left or right as a whole.

Plate Voltage field Adjusts the total voltage difference between the plates, which

determines the angle of stream deflection.

68 BD FACSAria User’s Guide

Table 2-2 Side Stream window controls

Control Description

2nd, 3rd, 4th Drop
fields

Phase field Adjusts the phase between drop generation and charging of the

Apply a correction factor for the drop charge as a percentage of
the previous drop, from –100% to 100%.

droplets from 0–360°.

The selected value is sent to both the drop-charging electrode
and the drop strobe. In general, the Phase never needs
adjusting; you can keep the default value of zero.

Drop Delay Overview

The BD FACSAria cytometer includes integrated Accudrop technology to assist
in setting an accurate drop delay value. Accudrop components consist of the
following:

• a diode laser, mounted to the left of the sort block

• a camera that provides an image of the side streams

• an emission filter for viewing the fluorescence from Accudrop beads

The emission filter is installed in front of the lower camera and can be moved in
and out by clicking the Optical Filter control. When the button is green ( ), the
filter is out. This position is used to view the center and side streams.

Click the button to move the filter in front of the camera when you are
determining the drop delay; the button changes to red ( ). To determine the
drop delay, the streams are illuminated by the diode laser just below the point of
deflection. Specialized fluorescent particles (Accudrop beads) can be viewed in
the center and left side streams as the delay is adjusted. The best delay yields the
most particles in the left stream and the fewest in the center stream. See
Determining the Drop Delay on page 158 for more information.

Chapter 2: Theory of Operation 69

Drop Charging

Drops are charged when an event is detected and meets the defined sorting
criteria, as specified in the Sort Layout window.

The Sort Layout window contains all sorting instructions and controls
(Figure 2-16). 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. See Sort Layout on page 96 for more information about the Sort Layout
window.

Figure 2-16 Sort layout for a four-way sort

Target events are identified by drawing gates around populations of interest in
plots. The Sort Layout window specifies which gated population should be sorted
into each sort collection tube or spot in a plate or on a slide. During sorting,
when an event is identified within one of the sort gates, the drop containing the
particle of interest is charged via the stream-charging wire in the nozzle.

stream-charging wire

70 BD FACSAria User’s Guide

The amount and type of charge determines where the drop will be sorted. For
example, in a four-way sort, drops with the most charge will be deflected into the
outer streams, while drops with less charge will be deflected into the inner
streams.

Conflict Resolution During Sorting

During sorting, the cytometer deflects drops based on the characteristics of the
particles in each drop and where the user wants to deflect them. Conflicts can
occur depending on the type of target particle, where the particle is located
within a drop, or whether the drop is free of contaminating particles.
BD FACSDiva software 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.

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 2-17 on page 72.

Chapter 2: Theory of Operation 71

Figure 2-17 Target particle within a Yield Mask of 16

trailing drop: sorted drop being interrogated leading drop: not sorted

Yield Mask

Yield Mask

target particle

If the Yield Mask were set to 8 for the same target particle, the target particle
would fall outside of the Yield Mask; therefore, no additional drops would be
sorted. See Figure 2-18.

Figure 2-18 Target particle outside a Yield Mask of 8

trailing drop: not sorted drop being interrogated leading drop: not sorted

Yield Mask

target particle

Yield Mask

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 0–32 will sort either one or two drops.

Yield Masks cannot be used in conjunction with Phase Masks. Therefore, when
the Yield Mask is greater than zero, the Phase Mask automatically reverts to
zero.

72 BD FACSAria User’s Guide

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 2-19).

Figure 2-19 Non-target particle within a Purity Mask of 16

trailing drop leading drop

drop being interrogated

Purity Mask

(not sorted)

Purity Mask

non-target particle

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 2-20.

Figure 2-20 Non-target particle outside a Purity Mask of 8

trailing drop leading drop

Purity Mask

drop being interrogated

(sorted)

Purity Mask

non-target particle

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. If the purity
mask is set to zero, a droplet containing the event of interest will be sorted
regardless of contaminating particles.

Chapter 2: Theory of Operation 73

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 2-21).

Figure 2-21 Sorted and unsorted drop with Phase Mask of 16

trailing drop (drop sorted) leading drop

Phase Mask

trailing drop (drop not sorted) leading drop

Phase Mask

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 2-22).

Figure 2-22 Sorted drop with Phase Mask of 8

trailing drop leading drop

(drop sorted)

Phase Mask

 Tip BD recommends using a Phase Mask of at least 8 when sorting single cells.

74 BD FACSAria User’s Guide

Phase Masks cannot be used in conjunction with Yield Masks. Therefore, 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, six Sort
Precision modes are already defined—Purity, 4-Way Purity, Yield, Single Cell,
Initial, and Fine Tune.

Precision Mode

4-Way

Purity

Yield Mask320320320

Purity Mask 32 32 0 32 0 0

Phase Mask 0 0 0 16 0 0

Single Cell

⌧

Purity Yield

Single

Cell Initial

Fine

Tune

• In Purity mode, the Yield Mask is set to the maximum to obtain the
greatest number of particles. Because the Purity Mask is also set to the
maximum, only drops free of contaminating particles will be sorted.
Sorting in Purity mode results in a sorted sample that is highly pure, at the
expense of recovery and yield.

• In 4-Way Purity mode, the Purity Mask is set to the maximum, so only
drops free of contaminating particles will be sorted. The Yield Mask is set
to zero to ensure that residual charges from adjoining drops do not degrade

Chapter 2: Theory of Operation 75

the quality of side streams. The 4-Way Purity mode is recommended for
four-way sorting where precise deflection is required.

• In Yield mode, only the Yield Mask is used at its maximum value. Recovery
and yield are optimized at the expense of purity.

• In Single Cell mode, the Purity Mask is set to the maximum, so only drops
free of contaminating particles will be sorted. The Phase Mask is set to 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.

 Tip 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.

The remaining modes are used mainly during drop delay determination. Initial
mode is equivalent to Yield mode, but it is named differently as a reminder to use
this as the initial mode when using Accudrop to set the drop delay.

• In Initial mode, only the Yield Mask is used at its maximum value.
Recovery and yield are optimized at the expense of purity.

• In Fine Tune mode, all masks are set to zero to deflect the maximum
number of drops. This mode is used to fine-tune the drop delay value. See
Determining the Drop Delay on page 158 for more information.

76 BD FACSAria User’s Guide

Defining New Precision Modes

Default Precision modes cannot be edited or deleted. However, you can create
new modes and then choose them from the Precision Mode drop-down menu.

1 Select Sort > Sort Precision and click Add.

The current sort mode is duplicated and the Mask fields are enabled.

2 (Optional) Change the name of the mode in the Precision Mode field.

3 Enter values for the Yield, Purity, and Phase Masks.

4 Select the Single Cell checkbox, if needed.

5 Click Close.

The new mode is added to the Precision Mode drop-down menu.

To delete a mode, choose it from the drop-down menu and then click Delete.

Chapter 2: Theory of Operation 77

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3

Using BD FACSDiva Software

All BD FACSAria cytometer functions are controlled using BD FACSDiva
software. This chapter provides a general overview of the workspace components
and describes software controls that are unique to the BD FACSAria cytometer.
For an in-depth description of software components not described in this chapter,
refer to the BD FACSDiva Software Reference Manual.

The following topics are covered in this chapter:

• Workspace Components on page 80

• Cytometer Controls on page 81

• Sorting Controls on page 92

• Templates on page 105

79

Workspace Components

When you start BD FACSDiva software, the workspace appears (Figure 3-1). For
a general overview of the workspace and to get started using the software, refer
to Getting Started with BD FACSDiva Software. When running BD FACSDiva
with the BD FACSAria, two additional windows can be displayed by clicking the
Sorting button on the Workspace toolbar. See Sorting Controls on page 92 for a
description.

Figure 3-1 BD FACSDiva workspace

80 BD FACSAria User’s Guide

Cytometer Controls

Most BD FACSAria-specific cytometer controls are
accessed through the Cytometer menu. Controls on the
Cytometer menu are described in the following sections:

• Fluidics Controls on page 81

• Cytometer Configuration on page 86

• Cytometer Status Report on page 89

The Cytometer Details and the Catalogs menu commands
are described in the BD FACSDiva Software Reference
Manual. Additional cytometer controls are located in the
Acquisition Dashboard. See Acquisition Controls on page 91.

Fluidics Controls

Fluidics control of the BD FACSAria cytometer is completely automated by
BD FACSDiva software. The software contains pre-programmed fluidics
protocols that are activated by choosing the corresponding menu command from
the Cytometer menu. In addition, fluidics level indicators are available in the
Cytometer window. See Fluidics Level Indicators on page 84.

Fluidics Startup

During fluidics startup, waste and sheath levels are verified and the fluidics
system is primed with sheath solution. The fluidics status is displayed at the
bottom of the main window.

See Cytometer Startup on page 108 for more information.

Chapter 3: Using BD FACSDiva Software 81

Fluidics Shutdown

Fluidics shutdown removes sheath from the lines and plenum and replaces it with
deionized (DI) water, and cleans the cuvette with a cleaning solution. See Fluidics
Shutdown on page 178 for more details.

Cleaning Modes

BD FACSDiva software contains pre-programmed cleaning modes that are
activated by choosing the corresponding menu command from the Cytometer >
Cleaning Modes menu. See Internal Cleaning on page 181 for more information.

Sheath Pressure

The sheath pressure determines how quickly particles pass through the laser
beam. Select Cytometer > Sheath Pressure to access a dialog box where you can
view the current sheath pressure.

82 BD FACSAria User’s Guide

Each sort setup option is optimized at a preset sheath pressure. If you
change the sheath pressure, a multitude of other values will be affected,
including the drop drive frequency, drop charge levels, laser delay, area
scaling factor, and other values. For proper cytometer operation, change the
sheath pressure by selecting an option from the Sort > Sort Setup menu. Do
not adjust the pressure using controls in the Sheath Pressure dialog box.
Note that the Sheath Pressure command is disabled when the Sweet Spot is
on.

Sample Agitation

Select Cytometer > Sample Agitation to specify the speed at which samples are
agitated. You can select from one of the specified values, or select Off to turn off
agitation.

Sample Temperature

Use the Sample Temperature command to set the temperature inside the sample
injection chamber. You can select from one of the specified values, or select Off to
turn off temperature control (Figure 3-2 on page 84).

Chapter 3: Using BD FACSDiva Software 83

Figure 3-2 Setting the sample temperature

cooling/heating
unit for sample
injection chamber

NOTE The cooling/heating unit is designed to maintain the temperature of a
sample tube. It is not designed to cool or heat the sample. It takes approximately
45 minutes to reach the required temperature inside the chamber, during which
time the chamber must be kept closed.

To cool or heat the chamber, install a sample tube and click Load. Click Stop
Acquiring to keep the chamber closed and stop running the sample. To maintain
the temperature, do not leave the chamber open for extended periods while
changing sample tubes.

Fluidics Level Indicators

BD FACSDiva software provides fluidics level indicators in the Cytometer
window (Figure 3-3 on page 85). The sheath and waste indicators provide an
approximate indication of the fluid levels in each tank. The DI, bleach, and
ethanol tank indicators appear full until the fluid level descends below 20% of
the tank capacity. When this occurs, the corresponding level indicator changes to
black.

84 BD FACSAria User’s Guide

Figure 3-3 Levels indicators

sheath

bleachDIwaste

ethanol

When the sheath is low or the waste is full while the stream is running, the
corresponding indicator turns red and the following warning message is
displayed every 5 minutes until the stream is turned off. If the warning message is
not dismissed, the system automatically turns off the stream after 15 minutes.

Do not dismiss the warning message without refilling the sheath or
emptying the waste. If you continue to run the cytometer after dismissing
the message, the waste container could overflow or the sheath container
could run dry.

If the cytometer is run until the sheath supply is depleted, air will
accumulate in the bubble filter. Purge the filter as described in Purging the
Bubble Filter on page 214. If you are unable to remove all of the air, replace
the bubble filter as described in Changing the Bubble Filter on page 203.

Chapter 3: Using BD FACSDiva Software 85

Cytometer Configuration

The menu selections shown in Figure 3-4 open the Cytometer Setup and Tracking
(CS&T) module in a separate window. The CS&T module enables you to
perform multiple functions related to cytometer configuration.

NOTE When the CS&T module is open, the FACSDiva software goes into a
holding mode. Once the CS&T module is closed, FACSDiva resumes operation.

Figure 3-4 Cytometer menu selections that open CS&T

Menu selections that open the
CS&T Module.

• View Configurations—Opens the Cytometer Configuration window within
the main CS&T window. See the following section for an overview of this
function.

• CST—Opens the CS&T module. See the Cytometer Setup and Tracking
Application Guide for complete information on using CS&T.

• Performance Tracking (LJ)—Opens the Performance Tracking feature
within the main CS&T window. See the Cytometer Setup and Tracking
Application Guide for complete information on using this feature.

86 BD FACSAria User’s Guide

Cytometer Configuration Window

The BD FACSAria cytometer is equipped with a specific set of lasers, filters, and
dichroic mirrors. The Cytometer Configuration window lets you define which
fluorochromes or cell parameters will be measured at each photomultiplier tube
(PMT) detector. Within this window, you can define parameters for an unlimited
number of fluorochromes, up to three lasers, and up to 15 detectors.

Select Cytometer > View Configurations to open the window shown in
Figure 3-5 on page 88. Click the Parameters tabs for a list of parameters. Click
the Filters and Mirrors tab for the list of optics.

The Cytometer Configuration window also displays these settings:

• Sheath pressure

• Nozzle size

• Window extension

•Sort mode

See the Cytometer Setup and Tracking Application Guide for complete
information on editing configurations, including adding parameters, mirrors, and
filters.

Chapter 3: Using BD FACSDiva Software 87

Figure 3-5 Example configuration for a three-laser system

Before you start any experiment, verify that the cytometer configuration contains
appropriate parameters for the samples you are running and that the cytometer
optics match the current configuration. See Application Options on page 250. If
needed, you can add or modify parameters in an existing configuration or define
a custom configuration, as described in Defining a Custom Configuration on
page 252.

For accurate data results, the cytometer optics must match the current
cytometer configuration.

88 BD FACSAria User’s Guide

Selections in the cytometer configuration determine which parameters are
available for your experiment. When more than one parameter is available for a
detector, the first parameter is listed by default. To select a different parameter,
click the name in the Parameters tab and select a different parameter from the
drop-down menu.

 Tip To ensure that your experiment’s parameters match those in the current

cytometer configuration, always record data with the same configuration.

Cytometer Status Report

The Cytometer Status Report provides a list of all cytometer settings at the time
the report was created. You must be connected to the cytometer to create the
report. In an open experiment, click to set the current tube pointer and select
Cytometer > Cytometer Status Report. The report is displayed in a separate
window with a menu bar above the report header. The header lists the cytometer
name, cytometer type, cytometer serial number, and the date and time the report
was prepared. See Figure 3-6 on page 90 for a sample report.

For a full description of the Cytometer Status Report, refer to the BD FACSDiva
Software Reference Manual. A BD FACSAria cytometer report includes the
following additional information.

Chapter 3: Using BD FACSDiva Software 89

Four types of information are displayed on the report: user access privileges,
cytometer information, cytometer settings, and sorting settings (for cytometers
equipped with sorting features). The report can be printed or exported.

• The User Access Privileges section lists access settings for the current user.

• The Cytometer Info section lists values for laser delay, area scaling, window
extension, FSC area scaling, sheath pressure, and sample flow rate.

• The Parameters section displays settings for the current acquisition tube.

• The Sort Settings section lists all sort setup values, along with the Plate
Voltage and Voltage Center values. If the Sweet Spot is off, Breakoff and
Gap values are shown. If the Sweet Spot is on, Drop 1 and target Gap
values are shown.

Figure 3-6 Cytometer Status Report

90 BD FACSAria User’s Guide

Acquisition Controls

Along with the controls described in the BD FACSDiva Software Reference
Manual, the following acquisition controls are available for the BD FACSAria

cytometer.

• Load—Lifts a tube into the sample injection chamber, starts sample
agitation (if agitation is turned on), and starts acquisition of the sample.

When a tube is already loaded, the button changes to Unload. Clicking
Unload stops acquisition of the sample, turns off the agitator, and lowers
the tube from the sample injection chamber.

NOTE The Load button is enabled only when the workstation is connected
to the cytometer, an experiment is open, the stream is turned on, and the
current tube pointer is set to a tube.

• Stop Acquiring—Stops sample acquisition without unloading the sample
tube.

The Acquire Data button functions only when a tube is loaded.

Chapter 3: Using BD FACSDiva Software 91

• Flow Rate—Controls sample flow rate, from 1.0–11.0 (10–120 µL/min).

Do one of the following to change the flow rate:

- Select the value in the field and enter a new value.

- Click the pointer in the slider bar and drag it to a new value.

- 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.

Sorting Controls

All sorting on the BD FACSAria cytometer is controlled by BD FACSDiva
software. Sorting controls are shown in Figure 3-7.

Figure 3-7 BD FACSDiva sorting controls

1

2

4

3

92 BD FACSAria User’s Guide

The Sort menu provides access to sort setup and sort precision modes, sort

1

layouts, sort reports, and sort devices used with the automated cell deposition
unit (ACDU). See Sort Menu on page 93.

The Sort Layout window designates which device will be used to collect sorted

2

particles and which particles will be sorted into each sort location. Sorting
controls and counters are also displayed in the window. See Sort Layout on
page 96.

The Breakoff window displays an image of the stream and contains controls to

3

adjust drop formation. See Drop Formation on page 63.

The Side Stream window displays an image of the side streams, and contains

4

controls to adjust electrical charges and the drop delay. See Side Stream
Formation on page 67.

Sort Menu

Choose commands in the Sort menu for the following.

• Sort Setup—Downloads the most recently used settings
for sorting. See Sort Setup on page 94.

• Sort Precision—Opens a dialog box where you can
choose or define a sort precision mode for handling
sorting conflicts. See Conflict Resolution During Sorting
on page 71.

• New Sort Layout—Opens the default 2-Tube Sort Layout window where
other sort layouts can be chosen. See Sort Layout on page 96. (Note that
clicking the Sort Layout button on the Browser toolbar performs the same
function.)

• Open Sort Layout—Opens 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.

Chapter 3: Using BD FACSDiva Software 93

• Home Device—Opens a dialog box containing commands to move the tray
support arm, either manually or to the home position. See Setting Up the
Stream on page 171.

• Custom Devices—Opens a dialog box where custom devices can be
defined. See Creating a Custom Device on page 174.

• Sort Report—Displays a report showing the sort settings, acquisition
counters, and sort layout information from the current sort. See Sort
Report on page 103.

Sort Setup

Sort setup values for four different nozzle sizes can be downloaded using the Sort
> Sort Setup command. Default settings are provided for each sorting option,
along with a custom setting for each option. Use the following guidelines to
determine which option to use. As a general rule, for optimal results when sorting
large or fragile cells, use a larger nozzle size and lower pressure. To increase
throughput and yield when sorting smaller or less fragile cells, use a smaller
nozzle size and higher pressure.

Sort Setup name Nozzle (micron) Default pressure (psi)

70 micron 70 70

85 micron

100 micron 100 20

130 micron

a. Contact your BD Sales representative for information on the availability of the

a

a

85 and 130 micron nozzles.

85 45

130 10

If you make changes to any of the settings, the changes are automatically saved
when you exit BD FACSDiva software or when you switch to a different sort
setup mode. When you restart, the most recently used set of values is restored.
Default settings are shown in Table 3-1 on page 95.

In addition, the sort setup menu has following menu items: 70 Custom, 85
Custom, 100 Custom, and 130 Custom. Initially, all of these new setups are exact

94 BD FACSAria User’s Guide

copies of the corresponding original setups with the same defaults. This will
provide users with the flexibility to create two sets of sort values (pressure for
example) for the same nozzle size.

Do not use a nozzle size that is different from the sort setup setting. The gap
values and sensitivity algorithms are optimized for nozzles and are not
suitable for other nozzle sizes. The system performance could be unstable.

Table 3-1 Default Sort Setup values

BD FACSDiva v6.1 Setting 70 micron 85 microna100 micron 130 micron

BD FACSDiva v6.0 Setting High Medium Low Custom

Sheath Pressure 70 45 20 10

Amplitude 60 32 12 24

Frequency 87 47 30 12

Drop 1 150 150 150 150

Gap (upper limit) 6 (14) 7 (17) 10 (21) 12 (21)

Attenuation Off Off Off Off

Drop Delay 47.00 30.00 27.00 16.00

Far left voltage 100 100 80 60

Left voltage 40 35 30 20

Right voltage 40 35 30 20

Far right voltage 100 100 80 60

Plate voltage 4,500 4,000 2,500 2,000

2nd Drop 20 20 10 0

a

3rd Drop 10 10 5 0

4th Drop 0 0 0 0

Laser Delay (blue) 0.00 0.00 0.00 0.00

Chapter 3: Using BD FACSDiva Software 95

Table 3-1 Default Sort Setup values (continued)

BD FACSDiva v6.1 Setting 70 micron 85 microna100 micron 130 micron

BD FACSDiva v6.0 Setting High Medium Low Custom

Laser Delay (red) -36.00 -54.00 -40.00 -82.00

Laser Delay (violet) 36.00 54.00 40.00 82.00

Area Scaling (blue) 1.40 1.20 1.40 0.75

Area Scaling (red) 1.00 0.75 1.00 0.50

Area Scaling (violet) 0.80 0.35 0.50 0.25

Window Extension 2.00 2.00 2.00 4.00

a. Contact your BD Sales representative for information on the availability of the 85 and 130 micron

nozzles.

a

Sort Layout

The Sort Layout window contains 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 global worksheets. Sort layouts are available for up to nine
default collection devices. Additional custom devices can be defined. See Creating
a Custom Device on page 174.

default collection
devices

96 BD FACSAria User’s Guide

Examples of sort layouts for different devices are shown in the following figures.
Instructions for setting up the sort layout can be found in Setting Up a Sort
Layout on page 98.

Figure 3-8 Sort layout for collection tubes (top) and for a 48-well plate (bottom)

collection

device

sort

counters

sorting

controls

Figure 3-9 Sort layout for a frosted slide

sort location
field for
far-right tube

sort location
field for
individual well

sort location field
for a spot on a slide

Chapter 3: Using BD FACSDiva Software 97

Setting Up a Sort Layout

Sort layouts can be added to tubes or global worksheets.

• Create tube-specific layouts if you are sorting different populations or using
different sort devices for each tube in the experiment.

• Create global worksheet–specific layouts if you are sorting the same
populations into the same sort device for all tubes in the experiment.

NOTE To create a tube-specific sort layout, make sure a normal worksheet
(white tab) is shown in the Worksheet window before you create the layout.

1 Select the icon for a tube or global worksheet in an open experiment, and

click the New Sort Layout button on the Browser toolbar ( ).

2 In the Sort Layout window, select 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 Select the sort precision mode from the Precision menu.

For more information, see Sort Precision Modes on page 75.

4 Enter the number of events to be sorted in the Target Events field.

Once entered, the number of events can be selected from the Target Events
menu. For continuous sorting, select Continuous from the Target Events
menu.

5 Choose Save Sort Reports option from drop-down menu. There are three

choices for saving the sort report:

• Save None—Sort Reports are not saved.

• Save All—Automatically saves sort report each time the sort is stopped.

98 BD FACSAria User’s Guide

• Ask User—Prompts the user each time the sort is stopped to select
whether or not to save the sort report. This is the default option.

This setting is saved with the sort layout.

6 Select the field(s) corresponding to the tube(s), well(s), or spot(s) where the

population will be sorted and select a defined population from the Add
menu.

After you click in a sort location field, a menu appears where you can select
to add, delete, or clear all populations in the field (Figure 3-10).

Figure 3-10 Adding populations to be sorted

After you add a population, the population and the number of target events
are added to the corresponding sort location field.

 Tip Select a row or column header to select all fields in that row or

column. When you add a population, it will also be added to all selected
fields at once.

7 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.

Chapter 3: Using BD FACSDiva Software 99

• For a two-tube layout, conflicts are sorted to the right.

• For a four-tube layout, conflicts for the Far Left tube are sorted to the
left, and conflicts for the Far Right tube are sorted to the right.

Editing a Sort Layout

• To change the number of events for any population, click in the Sort
Location field(s) containing the population, then select a number from the
Target Events menu or enter a new number.

• To remove a population from a sort location field, select the field, then
select the corresponding population from the Delete menu.

• To clear all populations from a field, select the field, then select Clear All.

Using Sorting Controls

Sorting controls appear at the bottom of the Sort Layout window. Use these
controls for the following functions.

Access Stage

Pause/Resume

100 BD FACSAria User’s Guide

Move Drawer