FACAAria Users Guide Rev A March 2003

© 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 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

iv

45

77

Workspace Components...

78

Instrument Controls...

79

Fluidics Controls...

80

Fluidics Level Indicators...

83

Instrument Configuration...

84

Instrument Status Report...

85

Acquisition Controls...

86

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...

100

Setting Up the Fluidics Cart...

102

Instrument Quality Control...

105

Setting Up the Experiment...

105

Verifying Area Scaling and Laser Delay...

112

Reusing the Experiment...

120

Sample Optimization Using Instrument Setup...

121

Creating the Experiment...

122

Adjusting the Voltages and Threshold...

124

Calculating Compensation...

127

Recording and Analyzing Data...

129

Setting Up the Acquisition Template...

129

Recording Data...

131

Analyzing Data...

132

Reusing the Analysis...

135

Saving the Analysis...

135

Chapter 5: Sorting

137

Setting Up for Sorting...

138

Changing the Nozzle...

140

Setting Up the Breakoff...

142

Setting Up for Bulk Sorting...

143

Calculating the Drop Delay...

145

Setting Up the Experiment...

145

Sorting Beads to Determine the Drop Delay...

146

Contents

v

Sorting...

149

Setting Up the Experiment...

149

Starting and Monitoring the Sort...

151

Setting Up for Sorting Into a Plate or Slide...

153

Installing the Sorting Hardware...

153

Setting Up the Stream...

155

Creating a Custom Device...

158

Chapter 6: DNA Analysis Criteria for DNA Experiments...

162

CEN Optimization...

163

Setting Up the Experiment...

164

Running CEN...

166

CTN Resolution...

168

Running CTN...

168

Running Test Samples...

170

Chapter 7: Shutdown and Maintenance

vi

161

171

Daily Shutdown...

172

Fluidics Shutdown...

172

External Cleaning...

174

Scheduled Maintenance...

177

Internal Cleaning...

178

Purging Filters...

191

Changing Fluid Filters...

192

Changing the Sample Lines...

192

Changing Air Filters...

196

Unscheduled Maintenance...

196

Cleaning a Nozzle...

197

Cleaning the Camera Optics...

199

Cleaning Optical Filters...

200

BD FACSAria User’s Guide

Chapter 8: Troubleshooting

201

Troubleshooting the Stream...

202

Fluidics Troubleshooting...

206

Electronics Troubleshooting...

207

Acquisition Troubleshooting...

208

Sorting Troubleshooting...

214

Appendix A: Supplies and Options

217

Instrument Supplies...

218

Optical Components...

218

Accessory Kit...

219

Other Replacement Parts...

221

Consumables...

222

Instrument Setup Particles...

222

Reagents...

223

Labware...

224

Instrument Options...

225

Application Options...

226

Two-Laser System...

226

Three-Laser System...

227

Changing Filters...

228

Defining a New Configuration...

230

Index

233

Contents

vii

viii

BD FACSAria User’s Guide

About This Guide This user’s guide contains the instructions necessary to operate and maintain your BD FACSAria™ flow cytometer. Because many instrument functions are controlled by BD FACSDiVa™ software, this guide also contains basic software information needed for instrument setup. For more detailed instructions on how to use the software, refer to the BD FACSDiVa Software User’s Guide. The BD FACSAria User’s Guide assumes you have a working knowledge of basic Microsoft® Windows® operation. If you are not familiar with the Windows operating system, refer to the documentation provided with your computer. New users of the BD FACSAria flow cytometer should read: •

Chapter 1 to become familiar with your instrument components

•

Chapter 2 to understand how the instrument works and to learn about the software components used to control different subsystems

•

Chapter 3 to see where software components are located

Instructions for routine acquisition, analysis, and sorting can be found in Chapters 4 and 5. For information about running DNA samples, see Chapter 6. Once you become familiar with routine operation and need only a quick reminder of the main steps, use the quick reference guide located in the front pocket of this user’s guide.

ix

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. The word accompanying the hazard symbol indicates the level of severity, as shown in Table 2. Text and keyboard conventions are shown in Table 3. Table 1 Hazard icons Symbol

  B

Hazard

Symbol

Hazard

Dangerous voltage

 

Highly flammable

Laser radiation

General

Biological risk

Table 2 Severity indicatorsa Indicator

Level of Severity

WARNING

The hazard or unsafe practice could result in severe injury or death.

CAUTION

The hazard or unsafe practice could result in minor injury.

NOTICE

The hazard or unsafe practice could result in a possibly dangerous situation; material could be damaged or data could be lost.

a. In compliance with the ANSI Z535.4 standard: Product Safety Signs and Labels

x

BD FACSAria User’s Guide

Table 3 Text and keyboard conventions Convention

 Tip

Use Highlights features or hints that can save time and prevent difficulties

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.

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

Read the section of the user’s guide specific to the operation you are performing.

•

See Chapter 8, Troubleshooting.

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

product name, part number, and serial number

•

any error messages

•

details of recent system performance

For instrument support from within the US, call (877) 232-8995, prompt #2-2. For support from within Canada, call (888) 259-0187.

About This Guide

xi

Customers outside the US and Canada, contact your local BD representative or distributor.

xii

BD FACSAria User’s Guide

Safety and Limitations The BD FACSAria™ flow cytometer is equipped with safety features for your protection. Operate the instrument only as directed in the user’s guide. Keep this safety information available for future reference and for new users.

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 (325–1,400 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.

xiii

Laser Classification Laser hazard levels depend on laser energy content and the wavelengths used. 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 FACSAria flow cytometer is a Class I laser product per 21 CFR Subchapter J and EN 60825-1:1994 + A2:2001. The lasers are fully contained within the instrument 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  WARNING

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 instrument any place where laser warning labels are attached (see Precaution Labels on page xvii).

 CAUTION Controls, adjustments, or performance of procedures other than those specified in the BD FACSAria User’s Guide can result in exposure to hazardous visible or ultraviolet laser radiation, or both.

 CAUTION

Keep all instrument doors closed during instrument operation. When operated under these conditions, the instrument poses no danger of exposure to hazardous laser radiation.

xiv

BD FACSAria User’s Guide

Electrical Safety  WARNING

Lethal electrical hazards are present in some lasers, particularly in laser power supplies. Many portions of the electrical system, including the printed circuit boards, are at a dangerous voltage level. To prevent shock injury, follow these guidelines. •

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.

•

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.

•

Do not perform instrument maintenance or service procedures except as specifically stated in the user’s guide.

 WARNING A 12,000-volt potential exists between the deflection plates when they are on. Contact with the charged plates results in serious electrical shock. Do not touch the deflection plates when the voltage warning light is illuminated, or when the software indicates that the plate voltage is on. The plates remain energized even when the sort block door is opened.

 CAUTION

The nozzle and the stream-charging wire conduct electrical charge during sorting. To prevent shock, do not touch the nozzle or the stream-charging wire when the voltage warning light is illuminated or any time during sorting.

Safety and Limitations

xv

Biological Safety B WARNING All biological specimens and materials coming into contact with them can transmit potentially fatal disease. To prevent exposure to biohazardous agents, follow 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) 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 daily or whenever the waste indicator shows the waste is getting full.

B WARNING

Cell sorters that use droplet generation methods, like the BD FACSAria, can produce aerosols around the sample stream. When acquiring biohazardous samples, follow universal precautions at all times. Keep the sort block door closed during sorting. If you need to access the sort block, turn off the stream before opening the door. For information on laboratory safety, refer to the following guidelines. NCCLS documents can be ordered online at www.nccls.org.

xvi

•

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 FACSAria User’s Guide

General Safety  CAUTION

Movement of mechanical parts within the instrument can pinch or injure your hands or fingers. To prevent injury by moving parts, follow these precautions. •

Keep your hands and clothing away from the sample injection chamber when a tube is loading or unloading. Do not place objects underneath the sample injection chamber.

•

Keep your hands away from the sort aspirator drawer during sorting. The drawer can close at any time without warning if a clog is detected during sorting.

•

When sorting into a plate, keep the sort collection chamber door closed during sorting. Do not place objects inside the sort collection chamber, and do not attempt to access the plate until sorting is complete. The microtiter tray stage can move suddenly during the sorting process. Sorting continues even when the sort collection chamber door is opened.

Precaution Labels The following precaution labels appear on the BD FACSAria flow cytometer or fluidics cart to indicate a potential hazard. Do not remove these labels. Use appropriate precaution to avoid injury by the indicated hazard. See the previous sections in this booklet for more information. Label

B

Waste

Location(s)

Potential Hazard

Waste tank, waste tank connectors on fluidics cart

Risk of exposure to biologically transmissible disease

Safety and Limitations

xvii

Label



Ethanol



Location(s)

Potential Hazard

Waste tank cap

Risk of exposure to biologically transmissible disease

Ethanol tank, ethanol tank connectors on fluidics cart

Highly flammable material

Ethanol tank cap

Highly flammable material

Deflection plates indicator light

Dangerous voltage

Sort block door where deflection plates can be accessed (inside and outside)

Dangerous voltage

Ethanol

 

Deflection Plates

xviii BD FACSAria User’s Guide

Label

Location(s)

Potential Hazard



On or near all removable covers and any place where the laser beam can emerge from the instrument

Risk of exposure to hazardous laser radiation

Behind sample injection chamber

Risk of crushing or pinching by moving parts

Floor of sort collection chamber

Risk of crushing or pinching by moving parts

Power input on fluidics cart

Risk of instrument damage if wrong voltage applieda

DANGER DANGER

VISIBLE AND/ORAND/OR INVISIBLEINVISIBLE LASER RADIATION WHEN VISIBLE LASER REMOVED. AVOID EYE REMOVED. OR SKIN EXPOSURE TO RADIATION WHEN AVOID OR SCATTERED RADIATION. EYEDIRECT OR SKIN EXPOSURE TO DIRECT

OR SCATTERED RADIATION. 95-10361-00

95-10361-00

! CAUTION: MOVING PARTS

!

Peel Here

! WARNING: Before applying power, configure the power input module to the supply voltage. Consult the user's guide for instructions.

a. NOTE: Voltage is set by your service engineer during installation.

Limitations This instrument is for Research Use Only. Not for use in diagnostic or therapeutic procedures.

Safety and Limitations

xix

xx

BD FACSAria User’s Guide