Becton Dickinson FACSCalibur Manual de usuario

FACSCalibur

™

System

User’s Guide

02-61760-02

Becton Dickinson

Immunocytometry Systems

2350 Qume Drive

San Jose, CA 95131-1807

Ordering Information (800) 223-8226

Customer Support Center

(800) 448-2347 (BDIS)

FAX (408) 954-2347 (BDIS)

Becton Dickinson

Canada, Inc.

2464 South Sheridan Way

Mississauga, Ontario

L5J 2M8

Canada

Tel (905) 822-4820

FAX (905) 822-2644

Becton Dickinson

European HQ

Denderstraat 24

B-9320 Erembodegem-Aalst

Belgium

Tel (32) 53-720211

FAX (32) 53-720450

Nippon Becton Dickinson

Company, Ltd.

DS Bldg

5-26, Akasaka 8-chome

Minato-ku, Tokyo 107

Japan

Tel (81) 3-5413-8251

FAX (81) 3-5413-8155

Becton Dickinson

Worldwide, Inc.

30 Tuas Avenue #2

Singapore, 2263

Tel (65) 861-0633

FAX (65) 860-1590

August, 1996

11-10823-02 Rev. A

FACSCalibur User’s Guide

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

the prior written permission of Becton Dickinson Immunocytometry Systems

(BDIS), 2350 Qume Drive, San Jose, CA 95131, United States of America.

Disclaimer

BDIS reserves the right to change its products and services at any time to incorporate

the latest technological developments. This guide is subject to change without notice.

BDIS welcomes customer input on corrections and suggestions for improvement.

Although this guide has been prepared with every precaution to ensure accuracy,

BDIS assumes no liability for any error or omission, nor for any damages resulting

from the application or use of this information.

Trademarks

FACS and Falcon are registered trademarks of Becton Dickinson and Company.

FACSCalibur, C

ELL

Quest, FACSComp, FACSConvert, CONSORT, FACSFlow,

CaliBRITE, SimulSET, Attractors, PAINT-A-GATE

PRO

, FACStation, and

FACSNet, are trademarks of Becton Dickinson and Company.

Macintosh, Apple, and the Apple logo are registered trademarks of

Apple Computer, Inc.

ModFit

is a trademark of Verity Software House, Inc.

Limitations

Please refer to the appropriate reagent package inserts and software user’s guides for

specific instructions and limitations on in vitro diagnostic use.

The Sorting option, the FL4 option, and the Cell Concentrator Module option are

for research use only.

Use of controls or adjustments or performance of procedures other than those

specified in this user’s guide may result in hazardous laser light exposure.

FACSCalibur System User’s Guide

Table of Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Safety and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix

Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Components of the Basic FACSCalibur System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.4 Options and Upgrades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 2 Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.1 FACSCalibur Instrument Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.2 Fluidics Drawer Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Filling the Sheath Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Emptying the Waste Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Priming the Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Leaving the FACSCalibur Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2.3 Optical System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.4 Electronics System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.5 FACStation Data Management System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Chapter 3 Instrument Setup for Acquisition of Samples . . . . . . . . . . . . . . . . . . . . 29

3.1 Accessing Instrument Controls in C

ELL

Quest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.2 Optimizing the Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.3 Saving the Instrument Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Chapter 4 FL4 Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.1 Optics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

4.2 Time-Delay Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.3 Dual Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4.4 Setting up the FACSCalibur Instrument for Four-Color Analysis . . . . . . . . . . . . . . . . . . . 59

Turning on the Red-Diode Lase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Setting Up the FL4 Parameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Chapter 5 Sorting Option . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Sorting with the FACSCalibur System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Choosing a Sort Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

5.1 Priming the Sort Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

5.2 Preparing Collection Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

5.3 Creating a Sort Gate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

5.4 Selecting a Sort Gate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

5.5 Using the Sort Counters Window. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

5.6 Sorting the Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

5.7 Ending Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

5.8 Recovering Sorted Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

5.9 Cleaning the Sort Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

5.10 Aseptic Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Chapter 6 Cell Concentrator Module Option . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

6.1 Cell Concentrator Module Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

6.2 Preparing the Cell Concentrator Module to Sort . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

6.3 Sorting with the Cell Concentrator Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Priming the Sort Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Determining Reference Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Sorting and Concentrating Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Recovering Sorted Cells from the Sort Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Removing Cells for Re-analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Cleaning the Sort Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

Cleaning the Concentrator Vessel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Chapter 7 Cleaning and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

7.1 Daily Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

7.2 Monthly Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

7.3 Periodic Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Changing the Sheath Filter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Cleaning the Air Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Changing the Bal Seal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Changing the Sample O-ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

iii

FACSCalibur System User’s Guide

Chapter 8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Appendix A Consumables and Service Information . . . . . . . . . . . . . . . . . . . . . . 163

Appendix B FACSCalibur Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

FACSCalibur System User’s Guide

Preface

FACSCalibur™ is the Becton Dickinson Immunocytometry Systems (BDIS)

modular benchtop flow cytometer designed for applications ranging from

routine clinical to advanced research. This modular system features advanced

capabilities, such as the Sorting and FL4 options in an easy-to-use system.

Integral to the FACSCalibur system is the FACStation Data Management system

featuring a Macintosh

computer and C

ELL

Quest™ software, a general purpose

acquisition and analysis software program designed specifically for BDIS flow

cytometers.

FACSComp™ instrument setup software is also included with the system. Use

FACSComp for daily FACSCalibur system quality control and setup.

Preface

How to Use This Guide

This user’s guide contains the instructions necessary to operate and maintain

your FACSCalibur flow cytometer. The information is presented in easy-to-

follow steps in

boldface type

followed by additional information that provides

more detail. Because many FACSCalibur functions are controlled by C

ELL

Quest

software, you will also find the basic software information necessary for

instrument setup. If you are not familiar with the Macintosh computer or with

ELL

Quest software, refer to the appropriate Macintosh user’s guide provided by

Apple Computer, Inc. and the

ELL

Quest Software User’s Guide.

Use the table of contents and index to locate instructions for specific procedures.

Use the Quick Reference Guide, located in the jacket pocket of this user’s guide,

when you become familiar with the system and procedures.

Here’s what you’ll find in this user’s guide:

• Safety and Limitations, following this section, contains important

information you’ll need to know before operating the FACSCalibur system.

• Chapter 1, Introduction, deﬁnes the FACSCalibur system, giving an overview

of the FACSCalibur instrument, the FACStation data management system

and the software that comes installed.

• Chapter 2, Getting Started, provides you with the instructions necessary for

starting up the FACSCalibur instrument and preparing it for use. Also in this

chapter are instructions for turning on the computer and starting the

software.

• Chapter 3, Instrument Setup for Acquisition of Samples, describes how to

access instrument controls using C

ELL

Quest™ software, how to optimize

and save instrument settings, and provides instructions for setting up the

FACSCalibur system to run samples and collect data for multicolor analysis.

• Chapter 4, FL4 Option, provides instructions necessary for setting up the

FACSCalibur system to run samples and collect data for 4-color analysis.

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