FACSAria II Users Guide Rev A Dec 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 Becton, Dickinson and Company. 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 Clorox is a registered trademark of The Clorox Company. Fluoresbrite is a registered trademark of Polysciences, Inc. JDS Uniphase is a trademark of the JDS Uniphase Corporation. Microsoft, Windows, and Excel 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. Teflon is a registered trademark of E. I. du Pont de Nemours and Company. Contrad is a registered trademark of Decon Labs, Inc. iFlex2000 is a trademark of Point Source, Ltd. Kimwipes is a registered trademark of Kimberly-Clark Corp. Lauda is a registered trademark of Brinkman Instruments, Inc. All other company and product names might be trademarks of the respective companies with which they are associated. 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.

Patents PerCP: US 4,876,190 APC-Cy7: US 5,714,386 BD FACS™ Accudrop: 6,372,506 Sweet Spot: 5,700,692

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. 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

643245

12/07

Initial release

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Contents Conventions...

xiv

Technical Assistance...

xv

Limitations...

xvi

Chapter 1: Cytometer Components

17

Fluidics Cart...

18

Containers and Connectors...

18

Connecting to an External Air Supply...

20

Power and Operation...

21

Flow Cytometer...

22

Fluidics Components...

23

Optics System...

33

Cytometer Electronics...

39

Emergency Stop Button...

40

Workstation...

41

Chapter 2: Theory of Operation

43

Fluid Movement...

44

Sheath Flow...

44

Sample Flow...

46

Signal Generation...

48

Light Scatter...

48

Fluorescent Signals...

49

Signal Detection...

50

v

Detector Arrays...

50

Filters...

51

Detectors...

56

Electronic Processing...

57

Pulse Parameters...

58

Laser Delay...

59

Sorting...

60

Drop Formation...

61

Side Stream Formation...

65

Drop Charging...

68

Conflict Resolution During Sorting...

69

Chapter 3: Using BD FACSDiva Software

77

Workspace Components...

78

Cytometer Controls...

79

Fluidics Controls...

79

Fluidics Level Indicators...

83

Cytometer Configuration...

84

Cytometer Status Report...

88

Custom Configurations...

90

Acquisition Controls...

97

Sorting Controls...

98

Sort Menu...

100

Sort Setup...

101

Sort Layout...

103

Sort Report...

110

Templates...

112

Chapter 4: Running Samples

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113

Cytometer Startup...

114

Performing Fluidics Startup...

115

BD FACSAria II User’s Guide

Draft #5.1, October 2007

Starting the Stream...

118

Setting Up the Breakoff...

119

Setting Up the Fluidics Cart...

122

Checking Cytometer Performance...

130

Preparing the CS&T Workspace...

130

Preparing the CS&T Beads...

133

Running a Performance Check...

134

Reviewing the Results...

134

Application Settings...

136

Creating Application Settings...

136

Data Collection...

144

Setting Up the Workspace...

144

Calculating Compensation...

148

Data Recording and Analysis...

151

Setting Up the Experiment...

152

Setting Up the Global Worksheet...

153

Recording Data...

155

Analyzing Data...

156

Performing a Batch Analysis...

159

Chapter 5: Sorting

161

Setting Up for Sorting...

162

Setting Up for Bulk Sorting...

164

Determining the Drop Delay – Manual Method...

166

Setting Up the Experiment...

167

Using Manual Drop Delay...

168

Determining the Drop Delay – Automatic Method...

171

Overview of Auto Drop Delay

...

171

Using Auto Drop Delay...

171

Sorting...

173

Setting Up the Experiment...

174

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Contents

vii

Starting and Monitoring the Sort...

176

Pausing and Resuming a Sort...

178

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

179

Installing the Sorting Hardware...

179

Setting Up the Stream...

181

Creating a Custom Device...

184

Chapter 6: Shutdown and Maintenance

viii

187

Daily Shutdown...

188

Clean Flow Cell...

188

Fluidics Shutdown...

190

External Cleaning...

193

Scheduled Maintenance...

194

Internal Cleaning...

194

Purging the Fluid Filters...

200

Purging the Sheath Filter...

200

Flow Cell Preventive Cleaning...

201

Changing Fluid Filters...

202

Changing the Sheath Filter...

203

Changing the Sample Lines...

204

Changing the Air Filters...

211

Changing the Sheath Tank Air Filter...

212

Checking the Fluidics Cart Drip Tray...

212

Unscheduled Maintenance...

213

Changing Nozzles...

214

Cleaning the Nozzle...

216

Closed-Loop Nozzle Maintenance...

218

Installing or Removing a Sample Line Filter...

220

Changing the Pinch Valve Tubing...

223

Cleaning the Camera Windows...

225

Removing the Deflection Plates...

228

BD FACSAria II User’s Guide

Draft #5.1, October 2007

Lubricating the Sample Injection Chamber O-Ring...

229

Using Custom Optical Filters...

230

Cleaning the Optical Filters...

231

Removing or Installing the FSC ND Filter...

232

Chapter 7: Troubleshooting

233

Troubleshooting the Stream...

234

Troubleshooting the Breakoff...

238

Sorting Troubleshooting...

239

Acquisition Troubleshooting...

244

Fluidics Troubleshooting...

251

Electronics Troubleshooting...

252

Chapter 8: Technical Specifications

253

Cytometer Specifications...

254

Environment...

254

Performance...

255

Emission Optics...

257

Fluidics Cart Specifications...

259

Appendix A: Supplies and Consumables

261

Cytometer Supplies...

262

Optical Components...

262

Accessory Kit...

264

Other Replacement Parts...

266

Consumables...

267

Cytometer Setup Particles...

267

Reagents...

268

Labware...

269

Appendix B: Near UV Laser Option

271

System Laser Configurations...

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Contents

272

ix

Specifications...

273

Safety...

273

Operation...

274

Selecting Optical Filters...

274

Creating a Custom Configuration...

275

Switching From the Violet to the Near UV Laser...

276

Switching Back to the Violet Laser...

277

Side Population Application Guidelines...

278

Instrument Setup...

278

Experiment Setup Tips...

278

Troubleshooting...

280

Appendix C: BD Aerosol Management Option

281

Option Components...

282

Evacuator...

282

ULPA Filter...

283

Operating the BD Aerosol Management Option...

284

Starting Up the Evacuator...

284

Setting Up for Sorting...

287

Opening the Sort Collection Chamber Door...

288

Turning Off the Evacuator...

289

Maintenance...

289

Replacing the ULPA Filter...

290

Replacing the Air Filter...

294

Troubleshooting...

296

Control Panel Troubleshooting...

296

Filter Flow Gauge Troubleshooting...

298

Specifications...

299

Appendix D: Temperature Control Option

301

Option Components...

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BD FACSAria II User’s Guide

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Draft #5.1, October 2007

Using the BD Temperature Control Option...

303

Setting Up the Water Bath...

303

Setting Up the Tube Holder...

305

Setting Up the ACDU Stage...

307

Starting Up the Water Bath...

309

Maintenance...

310

Tube Holders...

310

Recirculating the Water Tubing...

310

Specifications...

311

Appendix E: QC Using BD FACSDiva Software

313

Cytometer Quality Control Using BD FACSDiva Software...

314

Setting Up the Cytometer Configuration...

314

Preparing QC Particles...

316

Adjusting Area Scaling and Laser Delay...

316

Reusing the QC Experiment...

328

Tracking QC Results...

329

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Contents

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About This Guide This user’s guide contains the instructions necessary to operate and maintain your BD FACSAria™ II flow cytometer. Because many instrument functions are controlled by BD FACSDiva™ software, this guide also contains basic software information needed for instrument setup. To familiarize yourself with the software, do the tutorials in the Getting Started with BD FACSDiva Software guide. For detailed information on software features, refer to the BD FACSDiva Software Reference Manual. The BD FACSAria II 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 II flow cytometer should read: •

Chapter 1 to become familiar with 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. Once you become familiar with routine operation and need only a quick reminder of the main steps, use the quick reference guide provided with this user’s guide.

xiii

Conventions The following tables list conventions used throughout this guide. Table 1 lists the symbols that are used in this guide or on safety labels 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 instrument, they are in black and white throughout this user’s guide; their meaning remains unchanged.

Table 2 Text and keyboard conventions

xiv

Convention

Use

; Tip

Highlights features or hints that can save time and prevent difficulties

NOTE

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, “select File > Print” means to select 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 FACSAria II User’s Guide

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 7, 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. 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

Limitations This instrument is for Research Use Only. Not for use in diagnostic or therapeutic procedures. BD Biosciences is providing software without warranty of any kind on an as-is basis. The software and workstations are intended for running the instruments supplied by BD Biosciences. It is the responsibility of the buyer/user to ensure that all added electronic files including software and transport media are virus free. If the workstation is used for Internet access or purposes other than those specified by BD Biosciences, it is the buyer/user’s responsibility to install and maintain up-to-date virus protection software. BD Biosciences does not make any warranty with respect to the workstation remaining virus free after installation. BD Biosciences is not liable for any claims related to or resulting from the buyer/ user's failure to install and maintain virus protection.

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BD FACSAria II User’s Guide

1 Cytometer Components The BD FACSAria II flow cytometer is a high-speed fixed-alignment benchtop cell sorter. The cytometer can be operated at varied pressures and can acquire up to 70,000 events per second. With its fixed-optics design and digital electronics, the BD FACSAria II flow cytometer enables multicolor analysis of up to 13 fluorescent markers and two scatter parameters at a time. The BD FACSAria II system consists of three major components: a fluidics cart, a benchtop flow cytometer, and a workstation (see Figure 1-1 on page 18). Nearly all cytometer functions are operated from within BD FACSDiva software. For a description of the system components, see the following sections. For technical information about how the cytometer works, see Chapter 2. •

Fluidics Cart on page 18

•

Flow Cytometer on page 22

•

Workstation on page 41

17

Figure 1-1 BD FACSAria II cytometer components

Fluidics Cart A separate fluidics cart supplies sheath and cleaning fluids and collects waste from the cytometer. The self-contained fluidics cart supplies the required air pressure and vacuum, which eliminates the need for an external source (although the cart can be hooked up to an in-house air source, if one is available). The air pumps provide pressure from 5 to 75 psi to accommodate a variety of cell sorting applications. Air pressure is adjusted within BD FACSDiva software.

Containers and Connectors The fluidics cart holds a 10-L stainless steel sheath tank, a 5-L stainless steel ethanol shutdown tank, a 10-L waste container, and three 5-L auxiliary cleaning fluid containers (Figure 1-2).

18

BD FACSAria II User’s Guide

Figure 1-2 Fluidics cart containers

3 auxiliary cleaning fluid containers

sheath filter

waste container

sheath tank

3 fluid filters

ethanol shutdown tank

To prevent foaming, do not fill the containers with solutions containing a high concentration of detergent. The fluidics cart connects directly to the flow cytometer unit via a power cord, fluid hoses, serial communication cable, and air line (Figure 1-3 on page 20). Receptacles for the aerosol management and temperature control options are also located within the connection panel. The position of the fluidics cart is constrained only by the length of the connecting cables and hoses, which extend up to 9 feet (2.7 m). Typically, the cart is placed to the left or underneath the cytometer.

Chapter 1: Cytometer Components

19

Figure 1-3 Fluidics cart power and fluid line connectors on cytometer

Waste connections for temperature control option

AMO connection

Air In

Waste Air Out

Fluid In Waste

Serial communication cable

Connecting to an External Air Supply To connect the fluidics cart to an external air source, switch on the Auxiliary Air Supply and attach the external air line to the air input connector. The external air supply should provide 80–100 psi. The external air must be dust and oil-free. Figure 1-4 Connectors on fluidics cart external air supply switch circuit breaker voltage selector switch

external air input connector air pressure gauge

NOTE There is a pull-out drip tray under the connection area. Check the tray periodically for moisture. See page 212 for details.

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BD FACSAria II User’s Guide