BD FACSAria III User's Guide

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Transcript of BD FACSAria III User's Guide

  • bdbiosciences.com23-11654-01 Rev. 015/2012

    For Research Use Only

    Becton, Dickinson and CompanyBD BiosciencesSan Jose, CA 95131Tel 877.232.8995Fax [email protected]

    BD BiosciencesEuropean Customer SupportTel 32.2.400.98.95Fax [email protected]

    BD FACSAria III Users Guide

  • Copyrights

    2012, 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.

    Trademarks

    Cy is a trademark of Amersham Biosciences Corp. 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 08855-1327, USA.

    Microsoft, Windows, and Excel are registered trademarks of Microsoft Corporation.

    Teflon is a registered trademark of E.I. du Pont de Nemours and Company.

    SPHERO is a trademark of Spherotech, Inc.

    Eppendorf is a registered trademark of Eppendorf AG.

    Fluoresbrite is a registered trademark of Polysciences, Inc.

    Clorox is a registered trademark of The Clorox Company.

    Kimwipes is a registered trademark of Kimberly-Clark Corporation.

    Lauda is a registered trademark of Metrohm USA, Inc.

    Alexa Fluor, Cascade Blue, and Texas Red are registered trademarks and Pacific Blue is a trademark of Molecular Probes, Inc.

    Living Colors is a registered trademark of Clontech.

    Contrad is a registered trademark of Decon Labs, Inc.

    BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 2012

    Regulatory information

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

    Class 1 Laser Product.

    Patents

    APC-Cy7: US 5,714,386

  • FCC Information

    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.

    NOTICE: 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 le matriel brouilleur du Canada.

    History

    Revision Date Change Made

    23-11654-00 Rev. 01 7/2010 Initial release.

    23-11654-01 Rev. 01 5/2012 Revised for BD FACSDiva software version 7.0.

  • v

    Contents

    About This Guide xiii

    Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv

    Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

    Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xvi

    Chapter 1: Cytometer Components 17

    New Software Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

    BD FACSAria III Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

    New and Upgrade Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

    Fluidics Cart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

    Containers and Connectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

    Connecting to an External Air Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

    Power and Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

    Flow Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

    Fluidics Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

    Optics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

    Cytometer Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

    Emergency Stop Button . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

    Workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

    Chapter 2: Theory of Operation 47

    Fluid Movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

    Sheath Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

    Sample Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

  • vi BD FACSAria III Users Guide

    Signal Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

    Light Scatter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

    Fluorescent Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

    Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

    Detector Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

    Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

    Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

    Electronic Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

    Pulse Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

    Laser Delay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

    Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

    Drop Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

    Side Stream Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

    Drop Charging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

    Conflict Resolution During Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

    Chapter 3: Using BD FACSDiva Software 81

    Workspace Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

    Cytometer Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

    Fluidics Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

    Fluidics Level Indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

    Cytometer Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

    Cytometer Status Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

    Custom Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

    Acquisition Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

    Sorting Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

    Sort Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

    Sort Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

    Sort Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

    Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

  • Co