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Appendix B: Near UV Laser Option 279
6 Adjust the voltages so that the red blood cells are seen in the lower left
corner and the dead cells line up on the vertical line to the far right.
7 The bulk of the rest of the cells can be centered. It should be possible to
identify a major G
0
/G
1
population with S/G
2
.
8 Collect a file of 50,000 to 100,000 of live gated events.
SP prevalence is ~0.02 to 0.05% of total.
9 If sorting, sort into tubes/plate containing small volume of DMEM
containing 10% FBS and 10 mmol HEPES. Keep sample chilled at 4
°C.
; Tip Sort rare event samples into polypropylene tubes. Avoid polystyrene
tubes.
Figure B-5 Typical plot from side population experiment
- BD FACSAria II 1
- User’s Guide 1
- FCC Information 3
- Contents 5
- About This Guide 13
- Conventions 14
- Technical Assistance 15
- Limitations 16
- Cytometer Components 17
- Fluidics Cart 18
- Power and Operation 21
- Flow Cytometer 22
- Fluidics Components 23
- Sample Injection Chamber 25
- Tube Holders 26
- Cuvette Flow Cell 27
- Figure 1-11 Nozzle 28
- Sort Block 29
- Deflection Plates 30
- Aspirator Drawer 30
- Aerosol Management 31
- Sort Collection Chamber 32
- Optics System 33
- Collection Optics 35
- Detectors 36
- Stream-Viewing Optics 38
- Cytometer Electronics 39
- Emergency Stop Button 40
- Workstation 41
- Theory of Operation 43
- Fluid Movement 44
- Sample Flow 46
- Hydrodynamic Focusing 47
- Signal Generation 48
- Fluorescent Signals 49
- Signal Detection 50
- Longpass Filters 52
- Bandpass Filters 53
- % transmission 54
- BP 500/50 filter 54
- DF 500/50 filter 54
- Neutral Density Filters 55
- Electronic Processing 57
- Pulse Parameters 58
- Laser Delay 59
- Drop Formation 61
- Breakoff Window 62
- Side Stream Formation 65
- Drop Delay Overview 67
- Drop Charging 68
- Yield Mask 70
- Purity Mask 71
- Phase Mask 72
- Sort Precision Modes 73
- Defining New Precision Modes 75
- Using BD FACSDiva Software 77
- Workspace Components 78
- Cytometer Controls 79
- Change Sample Filter 80
- Cleaning Modes 80
- Sheath Pressure 80
- Sample Agitation 81
- Sample Temperature 82
- Fluidics Level Indicators 83
- Cytometer Configuration 84
- Cytometer Status Report 88
- Custom Configurations 90
- Copying a Base Configuration 92
- Select the Sort Setup that 95
- Configuration Mismatch Dialog 96
- Acquisition Controls 97
- Sorting Controls 98
- Sort Menu 100
- Sort Setup 101
- Sort Layout 103
- Setting Up a Sort Layout 105
- Editing a Sort Layout 107
- Using Sorting Controls 107
- Using Counters 108
- Monitoring a Sort 109
- Sort Report 110
- Templates 112
- Running Samples 113
- Cytometer Startup 114
- Performing Fluidics Startup 115
- Starting the Stream 118
- Setting Up the Breakoff 119
- Setting Up the Fluidics Cart 122
- Refilling the Sheath Tank 123
- ATTENTION 124
- Emptying the Waste Container 127
- Waste (A) 129
- Preparing the CS&T Beads 133
- Running a Performance Check 134
- Reviewing the Results 134
- Application Settings 136
- Adjusting Area Scaling 137
- Figure 4-14 FSC and SSC Plot 138
- Optimizing PMT Voltages 141
- Saving Application Settings 143
- Data Collection 144
- Calculating Compensation 148
- Data Recording and Analysis 151
- Setting Up the Experiment 152
- Recording Data 155
- Analyzing Data 156
- Performing a Batch Analysis 159
- Setting Up for Sorting 162
- Setting Up for Bulk Sorting 164
- Using Manual Drop Delay 168
- Overview of Auto Drop Delay 171
- Using Auto Drop Delay 171
- Pausing and Resuming a Sort 178
- Setting Up the Stream 181
- Test Sort button 182
- Creating a Custom Device 184
- Deleting a Custom Device 186
- Shutdown and Maintenance 187
- Daily Shutdown 188
- Fluidics Shutdown 190
- External Cleaning 193
- Scheduled Maintenance 194
- Sample Line Backflush 195
- Prime After Tank Refill 196
- Prepare for Aseptic Sort 197
- DI water line 198
- DI water port 198
- Purging the Fluid Filters 200
- Purging the Sheath Filter 200
- Changing Fluid Filters 202
- Changing the Sheath Filter 203
- Changing the Sample Lines 204
- Figure 6-12 Ferrule tool 207
- Pinch Valve End 208
- Flow Cell End 209
- Changing the Air Filters 211
- Unscheduled Maintenance 213
- Changing Nozzles 214
- Cleaning the Nozzle 216
- Cleaning the Camera Windows 225
- Upper Camera Window 226
- Apply lubricant to outside 229
- Using Custom Optical Filters 230
- Cleaning the Optical Filters 231
- FSC ND filter 232
- Set screw 232
- Nozzle holder 232
- Troubleshooting 233
- Troubleshooting the Stream 234
- Troubleshooting the Breakoff 238
- Sorting Troubleshooting 239
- Acquisition Troubleshooting 244
- Fluidics Troubleshooting 251
- Electronics Troubleshooting 252
- Technical Specifications 253
- Cytometer Specifications 254
- Performance 255
- Sort Performance 256
- Emission Optics 257
- Excitation Optics 257
- Detector Arrays 258
- Fluidics Cart Specifications 259
- Appendix A 261
- Supplies and Consumables 261
- Cytometer Supplies 262
- Accessory Kit 264
- Other Replacement Parts 266
- Consumables 267
- Reagents 268
- Appendix B 271
- Near UV Laser Option 271
- System Laser Configurations 272
- Specifications 273
- Operation 274
- Instrument Setup 278
- Experiment Setup Tips 278
- Appendix C 281
- BD Aerosol Management Option 281
- Option Components 282
- ULPA Filter 283
- Starting Up the Evacuator 284
- Maintenance 289
- Replacing the ULPA Filter 290
- Reset button 293
- Replacing the Air Filter 294
- Appendix D 301
- Temperature Control Option 301
- Setting Up the Water Bath 303
- Cooling OutCooling In 304
- Setting Up the Tube Holder 305
- Input tubing 306
- Output tubing 306
- Setting Up the ACDU Stage 307
- Starting Up the Water Bath 309
- Appendix E 313
- QC Using BD FACSDiva Software 313
- Software 314
- Preparing QC Particles 316
- Setting Up the QC Experiment 317
- Recording QC Data 325
- Figure E-6 Recorded QC data 327
- Reusing the QC Experiment 328
- Tracking QC Results 329
- Numerics 331
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