http://www.bdbioscie Part No. 333608 Rev. A March 2003

BD Biosciences 2350 Qume Drive San Jose, CA 95131-1807 USA Tel (877) 232-8995 Fax (408) 954-2347

BD FACSAria User’s Guide

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© 2003, Becton, Dickinson and Company. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, stored in retrieval systems, or translated into any language or computer language, in any form or by any means: electronic, mechanical, magnetic, optical, chemical, manual, or otherwise, without prior written permission from BD Biosciences.

The information in this guide is subject to change without notice. BD Biosciences reserves the right to change its products and services at any time to incorporate the latest technological developments. Although this guide has been prepared with every precaution to ensure accuracy, BD Biosciences assumes no liability for any errors or omissions, nor for any damages resulting from the application or use of this information. BD Biosciences welcomes customer input on corrections and suggestions for improvement.

BD, the BD logo, BD CaliBRITE, BD CellQuest, BD FACS, BD FACSAria, BD FACSCalibur, BD FACSConvert, BD FACSRinse, BD FACStation, BD FACSVantage, and BD Falcon, are trademarks of Becton, Dickinson and Company.

Texas Red is a trademark of Molecular Probes, Inc. Clorox is a registered trademark of The Clorox Company. Innova is a registered trademarks of COHERENT, INC. FlowJo is a trademark of Tree Star, Inc. Java is a trademark of Sun Microsystems, Inc in the US and other countries. Microsoft and Windows are registered trademarks of Microsoft Corporation. Q-tips is a registered trademark of Chesebrough-Pond’s Inc. Teflon is a registered trademark of E.I. du Pont de Nemours and Company. Windex is a registered trademark of the Drackett Products Company.

All other company and product names might be trademarks of the respective companies with which they are associated.

Guide written by Kim Gautho; edited by Joan Miller; produced by Pushpa MacFarlane.

Regulatory Information

For Research Use Only. Not for use in diagnostic procedures.

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

Contents

About This Guide ix

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

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

Safety and Limitations xiii

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

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

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

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

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

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

Precaution Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii

Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Chapter 1: BD FACSAria Instrument Components 21

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

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

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

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

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

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

Instrument Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

BD FACSAria Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

iii

Chapter 2: Theory of Operation 45

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

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

Sample Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Signal Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Fluorescent Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Electronic Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Pulse Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Laser Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Drop Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Side-Stream Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Particle Charging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Conflict Resolution During Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Chapter 3: Using BD FACSDiVa Software 77

Workspace Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Instrument Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Fluidics Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Fluidics Level Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Instrument Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Instrument Status Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Acquisition Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

iv BD FACSAria User’s Guide

Sorting Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Sort Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Sort Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Sort Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Sort Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Chapter 4: Running Samples 99

Starting Up the Instrument . . . . . . . . . . . . . . . . . . . . . . .