BD FACSAria User’s Guide - Stanford Shared FACS facs. Recording and Analysis . . . . . . . . ....

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Transcript of BD FACSAria User’s Guide - Stanford Shared FACS facs. Recording and Analysis . . . . . . . . ....

  • bdbiosciences.comPart No. 640760 Rev. AMay 2006

    BD Biosciences2350 Qume DriveSan Jose, CA 95131-1807USATel (877) 232-8995Fax (408) 954-2347

    BD FACSAriaUsers Guide

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  • 2006, 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 FACS, BD FACSAria, BD FACSDiva, BD FACSFlow, BD FACSRinse, BD Falcon, and BD Trucount are trademarks of Becton, Dickinson and Company.

    Clorox is a registered trademark of The Clorox Company. Fluoresbrite is a trademark of Polysciences, Inc. JDS Uniphase is a trademark of the JDS Uniphase Corporation. Kimwipe is a registered trademark of the Kimberley-Clark Corporation. Microsoft and Windows are registered trademarks of Microsoft Corporation. Sapphire is a trademark and Coherent is a registered trademark of Coherent, Inc. SPHERO is a trademark of Spherotech, Inc. Texas Red, Alexa Fluor, and Cascade Blue are registered trademarks and Pacific Blue is a trademark of Molecular Probes, Inc.

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

    FCC 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 users 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 numrique de la classe A respecte toutes les exigences du Rglement sur the matriel 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, adjustable nozzle, waste cap with baffle and disposable filtered cap, adjustable nozzle, running samples using BD FACSDiva software version 4.1

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

  • 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 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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

    Instrument Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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

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

    Instrument Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

    Instrument Status Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

    Acquisition Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

    iv BD FACSAria Users Guide

  • Sorting Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

    Sort Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

    Sort Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

    Sort Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

    Sort Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

    Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

    Chapter 4: Running Samples 103

    Instrument Startup . . . . . . . . .