Molecular Devices FLIPR Tetra User manual

FLIPR®Tetra
High Throughput Cellular Screening System
User Guide
0112-0109 H
December 2011

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0112-0109 H 3
Contents
Chapter 1: System Overview . . . . . . . . . . . . . . . . . . . . . 13
Fluorescence Mode. . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Luminescence Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . 14
Electrical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Minimum Space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 2: System Hardware Features . . . . . . . . . . . . . 17
Overview of FLIPR Tetra System Hardware Features . . . . 17
System Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Plate-Handling System . . . . . . . . . . . . . . . . . . . . . . . . . 18
Five-Position Stage. . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Instrument Status Panel . . . . . . . . . . . . . . . . . . . . . . . 21
Manual Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Robotic Integration. . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Observation Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Liquid-Handling System . . . . . . . . . . . . . . . . . . . . . . . . 25
Standard Pipettor Head. . . . . . . . . . . . . . . . . . . . . . . . 25
Cell Suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Pin Tool Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Compatible Plate Configurations. . . . . . . . . . . . . . . . . . 30
Tip and Pin Tool Loading . . . . . . . . . . . . . . . . . . . . . . . 31
Tip and Pin Tool Washing . . . . . . . . . . . . . . . . . . . . . . 31
Optical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
CCD Camera Options . . . . . . . . . . . . . . . . . . . . . . . . . 33
LED Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Emission Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Chiller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Computer System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Host Computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Embedded Computer . . . . . . . . . . . . . . . . . . . . . . . . . 39
Chapter 3: Startup and Shutdown . . . . . . . . . . . . . . . . . 41

Contents
4 0112-0109 H
Starting Up the System . . . . . . . . . . . . . . . . . . . . . . . . . 41
Shutting Down the System . . . . . . . . . . . . . . . . . . . . . . 42
Chapter 4: Software Installation . . . . . . . . . . . . . . . . . . 43
Installing ScreenWorks Software . . . . . . . . . . . . . . . . . . 43
Activating the ScreenWorks Peak Pro License. . . . . . . . . . 44
Online vs. Offline Installation . . . . . . . . . . . . . . . . . . . . . 45
Online (Instrument) Mode . . . . . . . . . . . . . . . . . . . . . . 45
Offline (Desktop) Mode . . . . . . . . . . . . . . . . . . . . . . . . 46
Uninstalling ScreenWorks Software. . . . . . . . . . . . . . . . . 46
Chapter 5: ScreenWorks Software Overview . . . . . . . . . 47
ScreenWorks Software Main Screen . . . . . . . . . . . . . . . . 47
Title Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Menu Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Menu Bar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
File Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
View Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Instrument Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Tools Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Window Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Instrument Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Instrument Status Tab . . . . . . . . . . . . . . . . . . . . . . . . 66
Instrument Configuration Tab . . . . . . . . . . . . . . . . . . . 68
Process Explorer Tab. . . . . . . . . . . . . . . . . . . . . . . . . . 69
Experiment Window . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Constructing Protocols Using FLIPR Tetra Processes . . . . 70
Settings Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Setup Read Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Assign Plate to Position . . . . . . . . . . . . . . . . . . . . . . . . 74
Data File Name. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Folders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Temperature Control. . . . . . . . . . . . . . . . . . . . . . . . . . 77
Auto Print Options . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Analysis Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Viewing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

FLIPR®Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 5
Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Exporting Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Batch Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Image Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Transfer Fluid Process . . . . . . . . . . . . . . . . . . . . . . . . 108
Aspirate Table (Standard Pipettor) . . . . . . . . . . . . . . . 109
Dispense Table (Standard Pipettor) . . . . . . . . . . . . . . 112
Aspirate Configuration (Pin Tool) . . . . . . . . . . . . . . . . 115
Dispense Configuration (Pin Tool) . . . . . . . . . . . . . . . 116
Mix Fluid Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Mix Fluid (Standard Pipettor) . . . . . . . . . . . . . . . . . . . 117
Mix Fluid (Pin Tool). . . . . . . . . . . . . . . . . . . . . . . . . . 119
Mix with TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Read Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Read. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Read with TF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Wash Tips or Pins Process . . . . . . . . . . . . . . . . . . . . . . 123
Wash Tips (Standard Pipettor) . . . . . . . . . . . . . . . . . . 124
Wash Pins (Pin Tool). . . . . . . . . . . . . . . . . . . . . . . . . 125
Blot Pins Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Pause Pipettor Process . . . . . . . . . . . . . . . . . . . . . . . . 126
Wash Cell Reservoir Process . . . . . . . . . . . . . . . . . . . . 127
Finish With Source . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Chapter 6: Exchanging Hardware . . . . . . . . . . . . . . . . 129
Exchanging Pipettor and Pin Tool Heads . . . . . . . . . . . . 129
Uninstalling a Pipettor or Pin Tool Head . . . . . . . . . . . 129
Installing the Pipettor Head . . . . . . . . . . . . . . . . . . . . 131
Uninstalling Wash Reservoir Top . . . . . . . . . . . . . . . . 133
Installing Wash Reservoir Top . . . . . . . . . . . . . . . . . . 134
Resetting FLIPR Tetra System after Changing Pipettor Heads
134
Exchanging the 1536 Tip Gasket . . . . . . . . . . . . . . . . . 135
Installing the Gasket . . . . . . . . . . . . . . . . . . . . . . . . 135
Removing the Tip Block and Gasket . . . . . . . . . . . . . . 136
Exchanging Pin Tools . . . . . . . . . . . . . . . . . . . . . . . . . 137
Loading and Unloading the Pin Tool . . . . . . . . . . . . . . 137
Exchanging LED Modules . . . . . . . . . . . . . . . . . . . . . . 138

Contents
6 0112-0109 H
Uninstalling LED Modules. . . . . . . . . . . . . . . . . . . . . . 138
Installing LED Modules . . . . . . . . . . . . . . . . . . . . . . . 139
Changing Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Uninstalling a Filter . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Installing an Emission Filter . . . . . . . . . . . . . . . . . . . . 143
Installation of Plate Hold-Down Devices . . . . . . . . . . . . 144
Selection of Appropriate Plate Hold-Down . . . . . . . . . . 144
Cell Reservoir. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Installing Cell Reservoir. . . . . . . . . . . . . . . . . . . . . . . 146
Uninstalling Cell Reservoir. . . . . . . . . . . . . . . . . . . . . 147
Chapter 7: Calibration and Signal Test. . . . . . . . . . . . . 149
Optical Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Adding a Read Plate to Plate Library . . . . . . . . . . . . . . 149
Recalibrating the Optics. . . . . . . . . . . . . . . . . . . . . . . 150
Running a Signal Test . . . . . . . . . . . . . . . . . . . . . . . . . 153
System Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Running a Plate Prior to an Experiment . . . . . . . . . . . . 154
Chapter 8: Running an Experiment . . . . . . . . . . . . . . . 157
Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Preparing Cells for Adherent Assays . . . . . . . . . . . . . . . 157
Location of Cells in the Plate . . . . . . . . . . . . . . . . . . . 157
Cell Densities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Cell Seeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Preparing Cells for Suspension Assays. . . . . . . . . . . . . . 159
Location of Cells in the Plate . . . . . . . . . . . . . . . . . . . 159
Cell Densities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Cell Seeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Powering-Up the System . . . . . . . . . . . . . . . . . . . . . . . 159
Checking the System . . . . . . . . . . . . . . . . . . . . . . . . . 160
Running the Yellow Plate Signal Test. . . . . . . . . . . . . . 160
Dye-Loading the Cells for Fluorescence Assays. . . . . . . . 161
Loading Duration and Temperature. . . . . . . . . . . . . . . 161
Preparing a Source/Compound Plate . . . . . . . . . . . . . . . 162
Preparation Time for the Source Plate . . . . . . . . . . . . . 162
Recommended Source Plates . . . . . . . . . . . . . . . . . . . 162
Concentration of Compounds in the Source Plate . . . . . 162
Addition and Mixing of Compounds to the Cell Plate . . . 162

FLIPR®Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 7
Compound Plates for Suspension Assays. . . . . . . . . . . 163
Setting Up an Assay Protocol. . . . . . . . . . . . . . . . . . . . 164
Creating a Protocol File . . . . . . . . . . . . . . . . . . . . . . . 164
Optimizing the Optics and Fluid Dispensing . . . . . . . . . . 165
Optimizing the Hardware Settings . . . . . . . . . . . . . . . 165
Optimizing Optics Hardware . . . . . . . . . . . . . . . . . . . 165
Adjusting the Pipettor Height. . . . . . . . . . . . . . . . . . . 167
Adjusting the Fluid Dispensing Speed . . . . . . . . . . . . . 168
Optimizing Fluid Volume . . . . . . . . . . . . . . . . . . . . . . 168
Optimizing Pin Tool Delivery . . . . . . . . . . . . . . . . . . . 169
Optimizing Cell Delivery . . . . . . . . . . . . . . . . . . . . . . 169
Start the Assay Run . . . . . . . . . . . . . . . . . . . . . . . . . 169
FLIPR Calcium Assay Kit Protocol . . . . . . . . . . . . . . . . . 170
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Calcium Assay Kit Experimental Protocol. . . . . . . . . . . 172
Running the FLIPR Calcium Assay Kit . . . . . . . . . . . . . 174
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . 176
FLIPR Membrane Potential Assay Kit Protocol . . . . . . . . 177
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Cell Preparation for the FLIPR Membrane Potential Assay179
Dye Loading Using the FLIPR Membrane Potential Assay Kit
180
Running the FLIPR Membrane Potential Assay . . . . . . . 181
Troubleshooting the FLIPR Membrane Potential Assay Kit . .
183
Voltage Sensor Probes Assay Protocol . . . . . . . . . . . . . 184
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Cell Preparation for the voltage Sensor Probe Assay. . . 185
Running the Voltage Sensor Probe Assay . . . . . . . . . . 187
Luminescence Assay Protocol . . . . . . . . . . . . . . . . . . . 188
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . 188
Coelenterazine Loading For Adherent Assays . . . . . . . . 189
Coelenterazine Loading for Suspension Cell Assays . . . 190
Preparation of Cell Reservoir and Running the Assay . . 191
Optimizing an Assay . . . . . . . . . . . . . . . . . . . . . . . . . 194
Chapter 9: Troubleshooting . . . . . . . . . . . . . . . . . . . . . 197
Instrument Status Colors . . . . . . . . . . . . . . . . . . . . . . 197
Troubleshooting Start-Up . . . . . . . . . . . . . . . . . . . . . . 198

Contents
8 0112-0109 H
General Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . 200
Troubleshooting the Pipettor . . . . . . . . . . . . . . . . . . . . 205
Troubleshooting the Optics . . . . . . . . . . . . . . . . . . . . . 210
Troubleshooting the Yellow Plate . . . . . . . . . . . . . . . . . 215
Troubleshooting the Tip Washer . . . . . . . . . . . . . . . . . . 216
Troubleshooting the Cell Reservoir . . . . . . . . . . . . . . . . 218
Troubleshooting Data . . . . . . . . . . . . . . . . . . . . . . . . . 218
Troubleshooting Robotic Integration . . . . . . . . . . . . . . . 219
Appendix A: Robotic Integration . . . . . . . . . . . . . . . . . 221
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Document Conventions . . . . . . . . . . . . . . . . . . . . . . . 221
Interface Versioning . . . . . . . . . . . . . . . . . . . . . . . . . 221
Instrument Overview . . . . . . . . . . . . . . . . . . . . . . . . . 222
Instrument Basic Function . . . . . . . . . . . . . . . . . . . . . 222
Instrument Hardware Introduction . . . . . . . . . . . . . . . 222
Instrument Layout . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Plate Layout. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Plate-Handling System . . . . . . . . . . . . . . . . . . . . . . . 225
Instrument Layout Terminology (i.e., Where’s the front?) . .
226
Robotic Plate Loading . . . . . . . . . . . . . . . . . . . . . . . . 226
Optics Access Door . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Washer placement . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Cell Suspension Placement. . . . . . . . . . . . . . . . . . . . . 228
Other Instrument Access Areas . . . . . . . . . . . . . . . . . 228
Monitor and Keyboard Placement . . . . . . . . . . . . . . . . 230
Required access areas. . . . . . . . . . . . . . . . . . . . . . . . 230
FLIPR Tetra System Control Architecture . . . . . . . . . . . . 233
General Description . . . . . . . . . . . . . . . . . . . . . . . . . 233
Manual Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Remote Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Communication Protocol and Address . . . . . . . . . . . . . 236
General Remote Mode Use . . . . . . . . . . . . . . . . . . . . . 236
Setting Up Protocols for Remote Control Use . . . . . . . . . 240
General Directions . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Settings Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Analysis Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

FLIPR®Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 9
Transfer Fluid Processes . . . . . . . . . . . . . . . . . . . . . . 243
Wash Tips Processes . . . . . . . . . . . . . . . . . . . . . . . . 244
Mix Fluid Processes . . . . . . . . . . . . . . . . . . . . . . . . . 244
Read Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Finish with Source Processes . . . . . . . . . . . . . . . . . . . 244
Wash Cell Reservoir Processes. . . . . . . . . . . . . . . . . . 244
Command Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Command Syntax. . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Version<CR>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Status<CR> . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Loadplate<TAB>Location, Last Plate[, BAR CODE] <CR> . .
248
Removeplate<TAB>location <CR>. . . . . . . . . . . . . . . 250
Openprotocol<TAB>File Name<CR> . . . . . . . . . . . . . 251
Findprotocols<TAB>Folder<CR> . . . . . . . . . . . . . . . . 251
Runexperiment<TAB>[Data File Name]<CR> . . . . . . . 252
Stopexperiment<CR>. . . . . . . . . . . . . . . . . . . . . . . . 253
Clearerror<CR> . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
Loadtips<CR> . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Unloadtips<CR> . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Cyclecameratemp<CR> . . . . . . . . . . . . . . . . . . . . . . 257
Tempcontrolonoff<TAB>Temp<CR> . . . . . . . . . . . . . 257
Washtips<TAB>Fluid Type, Wash Cycles, Volume/Stroke,
Aspirate Speed, Pump Speed, Strokes, Hold Time, Dispense
Speed<CR> . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
Configuration <CR> . . . . . . . . . . . . . . . . . . . . . . . . . 260
Statusex<CR>. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Cellflaskcontrol<TAB>Rate<CR> . . . . . . . . . . . . . . . . 264
Washcellreservoir<TAB>Fluid Type, Fill Speed, Drain
Destination, Drain Speed, Wash Cycles, Hold Time, Volume
Level<CR> . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Error Handling and Reporting . . . . . . . . . . . . . . . . . . . 267
Error Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Appendix A—Remote Interface Revision History. . . . . . . 270
V1.0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
V1.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
V1.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
V1.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

Contents
10 0112-0109 H
Appendix B—Bar Code Specifications . . . . . . . . . . . . . . 271
About Bar Codes. . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Bar Code Recommendations . . . . . . . . . . . . . . . . . . . 271
Bar Code Specifications . . . . . . . . . . . . . . . . . . . . . . . 272
Appendix B: Data Processing Algorithms . . . . . . . . . . . 273
Hypothetical Experiment . . . . . . . . . . . . . . . . . . . . . . . 273
Crosstalk Correction . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Determining Crosstalk Correction . . . . . . . . . . . . . . . . 274
Spatial Uniformity Correction . . . . . . . . . . . . . . . . . . . . 275
Determining Spatial Uniformity Correction . . . . . . . . . . 275
Negative Control Correction . . . . . . . . . . . . . . . . . . . . . 276
Determining Negative Control Correction. . . . . . . . . . . 277
Positive Control Scaling . . . . . . . . . . . . . . . . . . . . . . . . 278
Determining the Positive Control Scaling . . . . . . . . . . . 278
Subtract Bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
Determining Subtract Bias . . . . . . . . . . . . . . . . . . . . . 279
Response Over Baseline . . . . . . . . . . . . . . . . . . . . . . . 280
Determining Response Over Baseline Correction. . . . . . 280
Appendix C: Consumables and Accessories . . . . . . . . . 283
FLIPR Tetra System Accessories . . . . . . . . . . . . . . . . . . 283
Field Installations . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Pipettor Heads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Optics Consumables . . . . . . . . . . . . . . . . . . . . . . . . . 284
Pipetting Consumables . . . . . . . . . . . . . . . . . . . . . . . 285
Cell Reservoir Consumables . . . . . . . . . . . . . . . . . . . . 286
Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
96-Well Read Plates . . . . . . . . . . . . . . . . . . . . . . . . . 287
96-Well Read Plate Masks . . . . . . . . . . . . . . . . . . . . . 287
96-Well Source Plates . . . . . . . . . . . . . . . . . . . . . . . . 288
384-Well Read Plates . . . . . . . . . . . . . . . . . . . . . . . . 289
384-Well Source Plates . . . . . . . . . . . . . . . . . . . . . . . 290
1536-Well Read Plates . . . . . . . . . . . . . . . . . . . . . . . 290
1536-Well Source Plates . . . . . . . . . . . . . . . . . . . . . . 291
Source Reservoirs. . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Assays Performed on the FLIPR Tetra System . . . . . . . . 291
Calcium Flux Consumables. . . . . . . . . . . . . . . . . . . . . 291
FLIPR Membrane Potential Assay Kit Consumables . . . . 293

FLIPR®Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 11
Equipment and Supplies Suggested to Perform
Assays with Your FLIPR Tetra System. . . . . . . . . . . . . . 293
Appendix D: Using AquaMax Sterilant . . . . . . . . . . . . . 295
Principle of Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Materials Required but Not Provided. . . . . . . . . . . . . . . 295
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
Reagent Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . 296
Reagent Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
Warnings, Precautions and Limitations . . . . . . . . . . . . . 296
Appendix E: Decontamination Certificate . . . . . . . . . . 299
Procedure for Completing the Certificate. . . . . . . . . . . . 299
Appendix F: Electromagnetic Compatibility (EMC) . . . 301
REGULATORY INFORMATION FOR CANADA
(ICES/NMB-001:2006) . . . . . . . . . . . . . . . . . . . . . . . . 301
ISM EQUIPMENT CLASSIFICATION (Group 1, Class A) . . 301
INFORMATION FOR THE USER (FCC NOTICE) . . . . . . . . 301

Contents
12 0112-0109 H

0112-0109 H 13
1
System Overview
This chapter provides an overview of the FLIPR®Tetra High Throughput
Cellular Screening System requirements.
FLIPR®Tetra Systems are fluorescence- and luminescence-based
microplate readers with an integrated 1536-, 384- or 96-pipettor. They
perform rapid throughput cell-based assays while providing accurate
and precise kinetic data. Primary applications include intracellular
calcium mobilization and membrane potential. In addition, an expanded
choice of wavelengths enables you to utilize a broad range of
fluorescent dyes.
The FLIPR®Tetra System includes:
•Simultaneous 96-, 384- or 1536-well liquid or cell transfer
•Expanded wavelength support
•User-configurable pipettors and optics
•Agile internal plate handling
•Standard, (EMCCD) camera for fluorescence applications or
Aequorin, (ICCD) camera for fluorescence and luminescence
•Cell suspension option
•Slim platform with minimal facilities requirements.
An overhead pipettor delivers compound to all wells of the read plate
simultaneously. A protocol file configured in ScreenWorks Software—
the system-control and analysis program for FLIPR®Tetra System—
coordinates timing of compound delivery with multiple time-point
exposures so that the resulting sequence of data points spans
compound addition.
ScreenWorks Software displays relative light units versus time for all
1536-, 384- or 96-wells on the system’s monitor. Updates occur in
real-time as allowed by processing speed. Data can be exported as
relative light units over time (time sequence), or as a single value per
well (statistic). Export data files are in ASCII text file format for input
into spreadsheet or database programs.
Fluorescence Mode
In fluorescence mode the system’s LEDs illuminate the bottom of a
1536-, 384- or 96-well ‘read’ plate containing cells loaded with
fluorescent dye, and measure the fluorescence in each well. By taking a
sequence of measurements in conjunction with compound application,
changes in fluorescence emission characteristics due to the binding of
particular ions (for example,Ca2+, H+or Na+) can be tracked.

System Overview
14 0112-0109 H
Light-emitting diodes (LEDs) in the FLIPR®Tetra System produce light
at distinct wavelength ranges to excite the fluorescent dye that has
been added to the cells in the read plate wells. The entire well plate
bottom is illuminated. Fluorescent light emitted by the dye—again, for
the entire plate—passes through an emission filter before being
captured in a CCD camera, standard EMCCD or Aequorin ICCD.
Fluorescence is measured from each well independently, and converted
into a numerical value. The FLIPR®Tetra System can be configured with
two LED banks and three emission filters, allowing the software
configuration of up to four excitation/emission wavelength
combinations (‘read modes’). Thus, up to four different fluorescence
effects can be measured within a single experiment.
Luminescence Mode
The FLIPR®Tetra System also provides luminescence mode with the
Aequorin, ICCD camera option. The instrument has a light-tight
enclosure so that it can operate in luminescence mode and a
specialized high-sensitivity ICCD camera can be installed in place of the
standard EMCCD camera. The ICCD camera is mounted directly
beneath the read plate, so images are taken of the entire bottom of the
plate. For cell suspension experiments, an integrated Cell Reservoir
allows uniform cell suspension to be pipetted directly into the read
plate. From the 3 filter positions available on FLIPR®Tetra System, it is
recommended to have one open position, so no filter will be used
during luminescence assay.
As with fluorescence, luminescence is measured from each well
independently, and converted into a numerical value.
System Requirements
This section provides a brief overview of electrical, physical and
environmental requirements of the FLIPR®Tetra System. Please refer
to the FLIPR®Tetra System Pre-Installation Guide for full details.
Electrical
FLIPR®Tetra System consumes 5 A continuous and 9 A peak of 110 V
power and requires 90–240 VAC power source at 50–60 Hz which
equates to 2.5 A continuous and up to 4.5 A peak at 240 VAC/50 Hz.
The system is supplied with a power cord appropriate for the country it
was shipped to. Additional shared outlets are required for computer and
monitor. A power strip is acceptable for providing the additional outlets
for the computer and monitor.

FLIPR®Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 15
Minimum Space
System dimensions are as follows:
•Without Cell Suspension Module or TETRAcycler™:
approximately 39 inches wide 27 inches deep 70 inches high
(991 mm wide 686 mm deep 1780 mm high).
•With Cell Suspension Module and TETRAcycler: approximately
53 inches wide 27 inches deep 70 inches high (1346 mm
wide 686 mm deep 1780 mm high).
FLIPR®Tetra System is designed with rolling castors so it can be readily
moved to make necessary adjustments and perform maintenance.
Leveling feet are also installed on the lower instrument chassis. These
feet are typically used for stabilizing the instrument when integrated
with a robot, but can also be used to establish a uniform instrument
deck level in situations where the lab floor is not flat. When running an
experiment, please make sure the instrument’s feet are lowered and
leveled.
The computer and monitor are mounted to the right front side of the
instrument with the included clamp, requiring a minimum lab space of
73 inches (1.85 m) wide 82 inches (2.08 m) deep for maneuverability.
A chiller with dimensions 11 inches wide by 13 inches deep 13 inches
high (279 mm 330 mm 330 mm) is connected via a 3-foot (914
mm) long tube to the right side of the instrument. It can be placed
anywhere within that 3 foot radius as long as the user has access to the
on/off button on the chiller.
A minimum 25 inch (635 mm) square footprint for tip wash bottles is
required to the right side of the instrument.
The cabinet should have a user access space of 48 inches (1.22 m) in
front 24 inches
(610 mm) behind 10 inches (254 mm) to the left for servicing the
instrument.
WARNING! FLIPR®Tetra System can weigh as much as 860 lbs
(390 kg). Ensure adequate personnel are present when
installing or moving the system. Follow all necessary safety
precautions and use proper moving techniques.

System Overview
16 0112-0109 H

0112-0109 H 17
2
System Hardware Features
Overview of FLIPR Tetra System Hardware Features
The FLIPR®Tetra System consists of a cabinet 39” (965 mm) wide 27”
(686 mm) deep 70” (1780 mm) high, with a number of components,
including wash bottles, Cell Suspension module, chiller, host computer
and monitor, outside the cabinet.
The cabinet has two compartments, top and bottom, accessed by
manual doors on the front of the cabinet.
A ‘five-position stage’ located in the top compartment is where read
and source plates are positioned during an experiment. There are also
positions for tips and tip washing, as well as the Cell Suspension
reservoir.
The TETRAcycler™plate shuttle on the back wall of the top
compartment can be used for robotically controlled carriage of
compound or read plates and tips in and out of the cabinet during
experiments. Plates are delivered to and from a landing pad outside the
cabinet on the left hand side.
Mounted on the back wall of the top compartment, above the
TETRAcycler, is the pipettor. The pipettor transfers compounds from
source plates to the read plate, and accesses the tip loading and tip
washing positions. When the Cell Suspension option is installed, the
pipettor also transfers cells in suspension from the Cell Reservoir to the
read plate.
The bottom, ‘dry’, compartment, houses the FLIPR®Tetra System
optics and an embedded computer for control of basic system
functions. Two LED excitation modules, to the left and right, direct light
up onto the base of the read plate, in the five-position stage above.
Light emitted from the read plate passes down through emission filters
directly below the plate to the camera (either the Standard EMCCD
camera or the Aequorin ICCD camera).
The system computer—running ScreenWorks®Software, through which
all user interaction with the system occurs—is attached to the outside
right-hand side of the cabinet. Monitor and keyboard are on an
adjustable arm attached on the right-hand side of the cabinet front.
The Cell Suspension module, if installed, is also mounted on the lower
right side of the instrument. This external module keeps the cells in
suspension and is connected via internal tubing to a Cell Reservoir that
is installed in Position 4 (Source Plate 3). The cells are kept in
suspension via stirring and are pumped into the reservoir for transfer to
the read plate. Up to 4 additional fluid bottles can also be connected to
the reservoir for cleaning purposes.

System Hardware Features
18 0112-0109 H
Containers for tip washer fill fluid and waste are placed outside the
cabinet beneath the computer.
Further information on these subsystems is presented in the following
sections.
System Diagram
Figure 2-1 Diagram of the complete FLIPR®Tetra System.
Plate-Handling System
Five-Position Stage
For an experiment, read and source plates are placed in the
five-position stage in the upper compartment of the FLIPR®Tetra
System, where the pipettor is able to transfer compound between

FLIPR®Tetra High Throughput Cellular Screening System User Guide
0112-0109 H 19
them. Plates can be loaded manually through the upper compartment
door, prior to an experiment, or robotically, as the experiment proceeds
, using the TETRAcycler.
The five positions of the stage are, from left to right:
•Position 1: Tips and/or Source Plate 1
•Position 2: Source Plate 2
•Position 3: Read Plate
•Position 4: Cell Reservoir and/or Source Plate 3
•Position 5: Tip Washer
Figure 2-2 The five-position stage.
Positions 1, 2 and 4 take standard, low volume, deep well and reservoir
source addition plates.
Tip loading and unloading occurs in Position 1, but this position can
double as a source plate position once tips are removed.
Position 3 opens to the optics chamber below for excitation of
fluorophores in read plate wells and emission reading.
Position 4 can be used for the Cell Reservoir included with the Cell
Suspension option. A single Cell Reservoir is compatible with all of the

System Hardware Features
20 0112-0109 H
FLIPR®Tetra System pipettor heads. When the Cell Reservoir is not
present, this position can be used as a source plate position.
Positions 1–4 have a mechanical plate sensor to identify the presence
of plates, tips or reservoirs.
Robotic integration enables the TETRAcycler to exchange up to 12
source plates and tip racks, and one read plate, in an experiment.
A dedicated tip wash reservoir is located in Position 5 and should be
configured to match respective pipettor heads (96, 384 or 1536).
Appropriate tip wash reservoirs are included in the purchase of a
pipettor head. Specific hardware components associated with tip
washing are described in Tip Washing, page 31.
Plates and tip racks are registered with well A1 in the lower left-hand
corner using a plate indexer found in Positions 1–4. The indexers also
serve as mechanical sensors to detect plate or tip presence. If plates or
tips are not present in a Manual Mode experiment, but requested by
software, the instrument will stop and end the experiment. During
Remote Mode, the system notifies the SynchroMax™ET or third-party
plate-handler that no plate or tip container is present and will stop the
instrument until plates or tips are detected. It is then the responsibility
of the SynchroMax ET or third-party plate-handler to deliver plates or
tips to the system.
Temperature Regulation
Positions 1, 2 and 4, for source plates, have optional temperature
control. Temperature settings range from ambient +5 °C to 40 °C.
Equilibrium temperature may take approximately 15 minutes to reach
the set temperature.
Configure temperature regulation with the Temperature Control
ON/OFF toggle command in the Instrument > Manual Operation
menu or corresponding button.
Note: Sensors can only detect plate or tip container presence. They
cannot identify the type of plate or tips. It is the user’s responsibility to
ensure that the correct plates and tips are loaded into position.
Note: FLIPR®Tetra System does not have humidified air flow.
Temperature regulation is easier to maintain during robotic integration
as temperature loss is minimized when plates are passed through the