LSRII User's Guide Rev A June 2007

© 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 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, BD logo and all other trademarks are property of Becton, Dickinson and Company. © 2007 BD Texas Red™ and Pacific Blue™ are trademarks, and Cascade Blue® and Alexa Fluor® are registered trademarks of Molecular Probes, Inc. Sapphire and Radius are trademarks and Coherent is a registered trademark of COHERENT, INC. ALPHA is a trademark of Omega Optical, Inc. SPHERO is a trademark of Spherotech, Inc. Xcyte is a trademark of Lightwave Electronics. Microsoft and Windows are registered trademarks of Microsoft Corporation. Teflon is a registered trademark of E.I. du Pont de Nemours and Company. JDS Uniphase is a trademark of JDS Uniphase, Inc. ALPHA is a trademark of Omega Optical, Inc. All other company and product names might be trademarks of the respective companies with which they are associated.

Patents BD LSR II is covered by one or more of the following US patents and foreign equivalents: 4,745,285; 4,844,610; and 6,014,904. PerCP: US 4,876,190 APC-Cy7: US 5,714,386 BD FACS lysing solution: US 4,654,312; 4,902,613; and 5, 098,849

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

History Revision

Date

Change Made

334717 Rev. A

12/02

Initial release

338639 Rev. A

10/04

Updated software terminology and screen shots for BD FACSDiva software version 4.1

640752 Rev. A

5/06

Updated software terminology and screen shots for BD FACSDiva software version 5.0

642221 Rev A

6/07

Updated software terminology and screen shots for BD FACSDiva software version 6.0

Contents About This Guide

xi

Conventions...

xi

BD LSR II Documentation...

xii

BD LSR II Online Help...

xii

Printed Documentation...

xiii

Electronic Documentation...

xiv

Technical Assistance...

xv

Safety and Limitations

xvii

Laser Safety...

xvii

Laser Product Classification...

xviii

Precautions for Safe Operation...

xviii

Electrical Safety...

xix

Biological Safety...

xx

General Safety...

xxi

Symbols and Labels...

xxi

Limitations...

xxiii

Chapter 1: Introduction

25

Overview...

26

Components...

26

Power Switch...

26

Handles...

27

Control Panel...

27

v

Fluidics...

28

Sample Flow Rate Control...

28

Fluid Control...

28

Sample Injection Port...

29

Sheath and Waste Containers...

31

Optics...

32

Lasers...

33

Filters...

34

Detectors...

35

BD LSR II Workstation...

37

Chapter 2: Cytometer Setup Starting the Cytometer and Computer...

40

Setting Up the Optical Filters and Mirrors...

41

Filter and Mirror Configurations...

42

Changing Optical Filters or Mirrors...

43

Filter and Mirror Specifications...

44

Preparing Sheath and Waste Containers...

45

Preparing the Waste Container...

47

Preparing the Fluidics...

48

Removing Air Bubbles...

49

Priming the Fluidics...

50

Quality Control...

51

Chapter 3: Running Samples

vi

39

53

Optimizing Your Cytometer...

54

Preparing the Workspace...

55

Setting Up an Experiment...

58

Optimizing the Voltages and Threshold...

63

Calculating Compensation...

65

Recording and Analyzing Data...

67

BD LSR II User’s Guide

Preparing the Workspace...

68

Recording Data...

68

Analyzing Data...

71

Reusing the Analysis...

75

Saving the Analysis...

75

Chapter 4: Maintenance

77

Maintaining the Cytometer...

78

Daily Cleaning and Shutdown...

78

Daily Fluidics Cleaning...

78

Daily Shutdown...

80

Scheduled Maintenance...

80

System Flush...

80

Waste Management System Maintenance...

82

Periodic Maintenance...

85

Changing the Sheath Filter...

85

Changing the Bal Seal...

88

Changing the Sample Tube O-Ring...

90

Appendix A: Technical Overview

91

Fluidics...

92

Optics...

93

Light Scatter...

93

Fluorescence...

94

Optical Filters...

95

Compensation Theory...

99

Electronics...

102

Pulse Measurements...

104

Digital Electronics...

105

Threshold...

105

Laser Controls...

106

Contents

vii

Appendix B: Troubleshooting Cytometer Troubleshooting... Appendix C: Supplies and Consumables

108 117

QC Particles...

118

QC Cytometer Setup and Tracking Particles...

118

Reagents...

119

Equipment...

120

Appendix D: Standard Base Configuration

121

4-Blue 2-Violet 2-355 UV 2-Red Configuration...

122

Base Configuration...

123

Octagon and Trigon Maps...

124

Additional Optics...

128

Appendix E: Special Order Configurations

viii

107

133

Common Special Order Configurations...

134

6-Blue 0-Violet 0-UV 3-Red Configuration...

135

6-Blue 2-Violet 0-UV 3-Red Configuration...

136

6-Blue 0-Violet 2-UV 3-Red Configuration...

137

6-Blue 2-Violet 2-UV 3-Red Configuration...

139

6-Blue 6-Violet 0-UV 3-Red Configuration...

140

6-Blue 6-Violet 0-UV 4-Red Configuration...

142

6-Blue 6-Violet 2-UV 3-Red Configuration...

144

6-Blue 6-Violet 2-UV 4-Red Configuration...

146

Special Order Configuration Trigon and Octagon Maps...

148

6-Color Blue Octagon Default Configuration Map...

149

2-Color Violet Trigon Default Configuration Map...

150

6-Color Violet Octagon Default Configuration Map...

151

2-Color UV Trigon Default Configuration Map...

152

3-Color Red Trigon Default Configuration Map...

153

BD LSR II User’s Guide

4-Color Red Octagon Default Configuration Map... Appendix F: Setting Laser Delay

154 155

About Laser Delay...

156

Optimizing Laser Delay...

157

Index

159

Contents

ix

x

BD LSR II User’s Guide

About This Guide This guide describes the procedures necessary to operate and maintain your BD™ LSR II flow cytometer. Because many cytometer functions are controlled by BD FACSDiva™ software, this guide also contains information about software features required for basic cytometer setup and operation. For an annotated list of additional documentation for your BD LSR II flow cytometer, see BD LSR II Documentation on page xii.

Conventions The following tables list conventions used throughout this guide. Table 1 lists the symbols that are used to alert you to a potential hazard. Text and keyboard conventions are shown in Table 2. Table 1 Hazard symbolsa 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 cytometer, they are in black and white throughout this user’s guide; their meaning remains unchanged.

xi

Table 2 Text and keyboard conventions Convention

; Tip

Use 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.

>

The arrow indicates a menu choice. For example, “choose File > Print” means to choose Print from the File menu.

Ctrl-X

When used with key names, a dash means to press two keys simultaneously. For example, Ctrl-P means to hold down the Control key while pressing the letter p.

BD LSR II Documentation BD LSR II Online Help The online help installed with your BD FACSDiva software contains the same text as that in the documents listed below, enhanced with features like full text search and related topic links. Access BD LSR II online help from the Help menu of BD FACSDiva software. BD LSR II online help topics are compiled from the following sources:

xii

•

BD FACSDiva Software Reference Manual

•

BD LSR II User’s Guide

•

BD High Throughput Sampler User’s Guide

BD LSR II User’s Guide

Printed Documentation A printed copy of the following documents is distributed with the BD LSR II flow cytometer: •

BD LSR II User’s Guide describes procedures necessary to operate and maintain your BD LSR II flow cytometer. Because many cytometer functions are controlled by BD FACSDiva software, this guide also contains information about software features required for basic cytometer setup and operation.

•

BD LSR II Safety and Limitations booklet discusses the safety features of the BD LSR II flow cytometer. It lists precautions for the cytometer’s laser, electrical, and biological hazards, and states limitations of use.

•

BD LSR II Facility Requirement Guide contains specifications for:

-

cytometer weight and size

-

temperature and other environmental requirements

-

electrical requirements

•

Getting Started with BD FACSDiva Software contains tutorial exercises that familiarize you with key software procedures and concepts.

•

A printed copy of the BD High Throughput Sampler User’s Guide is distributed with the BD™ High Throughput Sampler (HTS) option. This document describes how to set up and operate the HTS. It also contains a description of BD FACSDiva software features specific to the HTS.

About This Guide

xiii

Electronic Documentation PDF versions of the following documents can be found on the BD FACSDiva software installation disk or on your computer hard drive:

xiv

•

The BD FACSDiva Software Reference Manual includes instructions or descriptions for installation and setup, workspace components, acquisition controls, analysis tools, and data management. It can be accessed from the BD FACSDiva Software Help menu (Help > Literature > Reference Manual), or by double-clicking the shortcut on the desktop. In addition, a printed copy can be requested from BD Biosciences.

•

Getting Started with BD FACSDiva Software can be accessed from the Help menu (Help > Literature > Getting Started Guide), or by doubleclicking the shortcut on the desktop.

•

The BD LSR II User’s Guide and BD High Throughput Sampler User’s Guide PDFs can be found on the BD FACSDiva software installation disk in the Cytometer User Guides folder.

•

The BD FACSDiva Option White Paper can be downloaded from the BD Biosciences website. This white paper contains an in-depth discussion of the digital electronics used in the BD LSR II cytometer.

BD LSR II User’s Guide

Technical Assistance For technical questions or assistance in solving a problem: •

Read sections of the documentation specific to the operation you are performing (see BD LSR II Documentation on page xii).

•

See Troubleshooting on page 107.

If additional assistance is required, contact your local BD Biosciences customer support representative or supplier. When contacting BD Biosciences, have the following information available: •

product name, part number, and serial number

•

version of BD FACSDiva software you are using

•

any error messages

•

details of recent system performance

For cytometer support from within the US, call (877) 232-8995. For support from within Canada, call (888) 259-0187. Customers outside the US and Canada, contact your local BD representative or distributor.

About This Guide

xv

xvi

BD LSR II User’s Guide

Safety and Limitations The BD LSR II flow cytometer and its accessories are equipped with safety features for your protection. Operate only as directed in the BD LSR II User’s Guide and the BD LSR II Safety and Limitations booklet. Do not perform cytometer 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 (325–1400 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.

xvii

Laser Product Classification Laser hazard levels depend on laser energy content and the wavelengths used. Therefore, it is impossible to apply common safety measures to all lasers. A numbered system is used to categorize lasers according to different hazard levels. The higher the classification number, the greater the potential hazard. The BD LSR II flow cytometer is a Class I (1) laser product per 21 CFR Subchapter J and IEC/EN 60825-1:1994 + A1:2003 + A2:2001. The lasers and the laser energy are fully contained within the cytometer 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 cytometer any place where laser warning labels are attached (see Symbols and Labels on page xxi). 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 cytometer doors closed during cytometer operation. When operated under these conditions, the cytometer poses no danger of exposure to hazardous laser radiation.

xviii BD LSR II User’s Guide

Electrical Safety Lethal electrical hazards can be present in all lasers, particularly in laser power supplies. Every portion of the electrical system, including the printed circuit boards, should be considered to be at a dangerous voltage level. Avoid potential shock by following these guidelines. •

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

•

Connect the equipment only to an approved power source. Do not use extension cords. Have an electrician immediately replace any damaged cords, plugs, or cables. Refer to the BD LSR II Facilities Requirement Guide for specific information.

•

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 a power transformer or conditioner to convert the local power source to meet the BD LSR II power requirements (120 V ±10%, 50/60 Hz). Contact your local BD office for further information.

Safety and Limitations

xix

Biological Safety All biological specimens and materials coming into contact with them are considered biohazardous. Avoid exposure to biohazardous material by following 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) for 30 minutes 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 frequently or whenever the waste management system alarms.

For information on laboratory safety, refer to the following guidelines. NCCLS documents can be ordered online at www.nccls.org.

xx

•

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 Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue; Approved Guideline. Wayne, PA: National Committee for Clinical Laboratory Standards, 1997. NCCLS document M29-A.

•

Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Wayne, PA: National Committee for Clinical Laboratory Standards; 1990. NCCLS document H18-A.

BD LSR II User’s Guide

General Safety The cytometer handles are for BD Biosciences authorized personnel only. Do not access them or attempt to lift the cytometer with them, or you could injure yourself. To avoid burns, do not touch the fan guards on the back of the cytometer. The fan guards could be hot during and after cytometer operation. Movement of mechanical parts within the cytometer can pinch or injure your hands or fingers. To prevent injury by moving parts, keep your hands and clothing away from the cytometer during operation.

Symbols and Labels The following symbols, warnings, or precaution labels appear on the BD LSR II flow cytometer or the waste and fluid tanks. Symbol

Ꮨ Ꮩ 

Meaning

Location(s)

Dangerous voltage

Rear cytometer panel near power receptacle

Laser radiation hazard

Near all removable covers and any place where the laser beam can emerge from the cytometer

Caution! Consult accompanying documents.

Near the cytometer handles

Safety and Limitations

xxi