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Chapter 4: Running Samples 151
Data Recording and Analysis
Once you have optimized the cytometer electronics for your sample type, you are
ready to record and analyze data.
Before you record data, we recommend that you gate out doublets in order to
record only singlet events. The Doublet Discrimination Gating template provides
gated plots for this purpose.
During analysis, recorded data is displayed in plots, and gates are used to define
populations of interest. BD FACSDiva software analyzes the gated data and
calculates statistics that you can print or export. With global worksheets, data
can be shown for a series of tubes on the same worksheet, manually or in an
automated batch analysis.
The following sections describe how to use BD FACSDiva software features to
record and analyze sample data.
- 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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