Molecular Devices FLIPR Tetra User Manual
FLIPR® Tetra
High Throughput Cellular Screening System
User Guide
0112-0109 H
December 2011
This document is provided to customers who have purchased Molecular Devices, LLC
(“Molecular Devices”) equipment, software, reagents, and consumables to use in the
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Product manufactured by Molecular Devices, LLC.
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© 2011 Molecular Devices, LLC.
All rights reserved.
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
0112-0109 H 3
Contents
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
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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
0112-0109 H 5
Contents
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
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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
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Contents
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
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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
0112-0109 H 9
Contents
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
10 0112-0109 H
FLIPR® Tetra High Throughput Cellular Screening System User Guide
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
0112-0109 H 11
Contents
12 0112-0109 H
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.
1
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.
0112-0109 H 13
System Overview
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.
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FLIPR® Tetra High Throughput Cellular Screening System User Guide
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.
0112-0109 H 15
System Overview
16 0112-0109 H
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.
2
0112-0109 H 17
System Hardware Features
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
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FLIPR® Tetra High Throughput Cellular Screening System User Guide
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
0112-0109 H 19
System Hardware Features
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.
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.
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: 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
20 0112-0109 H
FLIPR® Tetra High Throughput Cellular Screening System User Guide
Plates
FLIPR® Tetra System accepts 96-, 384- and 1536-well plates that
conform to the proposed ANSI standards submitted by the Society for
Biomolecular Sciences. A sample of suitable source and read plates is
provided in Appendix C: Consumables and Accessories on page 283.
Black walled, clear-bottomed read plates provide an optimal imaging
environment for fluorescence assays. These plates prevent signal
diffraction while allowing excitation and signal access. Black walled,
clear-bottomed plates or white walled plates can be used for
luminescence assays.
For 96-well read plates, an optional slit-shaped mask can be used to
minimize saturation and edge effects associated with these plates.
Simply place the mask over the read position. See Appendix C for types
of masks available.
No mask is required for 1536- and 384-well plates.
Instrument Status Panel
The Instrument Status panel, located next to the upper door handle,
indicates whether or not the instrument is running and safe to open. It
includes an emergency Interrupt button to stop any processes.
Figure 2-3 The Instrument Status panel.
0112-0109 H 21
System Hardware Features
The panel has two lights as well as the Interrupt button. From the top
of the panel these are:
• Assay Running (Locked)—Yellow light
• Assay Finished (Unlocked)—Green light
• The Interrupt button is an override button to halt all tasks, so
CAUTION! The Interrupt button immediately ends the experiment and
should only be used in emergencies. The system may need to be
reinitialized by selecting Reset from the Instrument menu prior to
resuming normal instrument function.
Manual Mode
In manual operation all assay components must be positioned in the
five-position stage by hand, through the upper manual door, prior to
running an experiment. Once the experiment starts no further plate or
tip changes can be made. If you need to exchange plates or tips during
an experiment then you must run the FLIPR® Tetra System in Remote
Mode, using the TETRAcycler to replace used tips or plates.
The FLIPR® Tetra System always starts in Manual Mode. Toggle
between manual and remote modes with the Set Manual Mode and
Set Remote Mode commands in the Instrument menu, or use the
software buttons available.
In Manual Mode, the TETRAcycler gripper parks itself on the platelanding pad.
®
The FLIPR
Tetra System is performing a task. The upper and
lower doors are locked and cannot be opened until the task
finishes or is halted using the Interrupt button.
No tasks are being run and it is safe to open the upper and
lower instrument doors.
you can access the instrument. If pressed the yellow light
flashes until the system has reached a safe state to open the
doors, when the green light comes on.
Note: The top compartment door should remain closed during normal
system operation. Do not operate the instrument if the door is open.
All system functions halt when the door is open.
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FLIPR® Tetra High Throughput Cellular Screening System User Guide
Robotic Integration
To increase the number of plates you can use in an experiment (and
minimize personnel requirements), use the TETRAcycler internal plate
handler coupled with the SynchroMax ET or a third-party plate handler
(for example, stacker system or robotic arm). One read and up to 12
source plates and tip racks can be shuttled in and out of the FLIPR®
Tetra System in one experiment with this method.
When using automated delivery the SynchroMax ET or third-party plate
handler delivers plates to, and picks them up from, the landing pad on
the outside left of the instrument, from where the TETRAcycler shuttles
them in and out of the read compartment. The shuttle door over the
landing pad opens and closes to maintain a light-tight environment
within the compartment.
During automated operation the SynchroMax ET or third-party plate
handler controls the FLIPR® Tetra System by sending instructions to
load protocols, run experiments, and retrieve plates from the landing
pad. These commands are executed immediately upon receipt by the
instrument. Persisting instrument settings cannot be made from the
remote controlling program—these must be configured in ScreenWorks
Software before control is passed to the plate-delivery system.
To pass control to the plate-delivery software select Set Remote Mode
in the Instrument menu in ScreenWorks Software. The FLIPR® Tetra
System remains in remote control until Set Manual Mode is pressed.
The third-party plate handler software communicates with the FLIPR®
Tetra System computer via the serial communication port using TCP/IP.
SynchroMax ET software is installed with ScreenWorks Software on the
FLIPR® Tetra System computer so it is able to communicate directly
with the instrument. See Appendix A: Robotic Integration on page 221
for remote control syntax.
TETRAcycler™
The TETRAcycler is a plate gripper that runs along the back wall of the
upper read compartment, giving it access to Positions 1–4 in the
five-position stage and the landing pad on the outer left-hand side of
the cabinet. It shifts source plates and tip containers between these
locations when under the control of the SynchroMax ET or a third-party
plate handler (see above).
The TETRAcycler carries standard, low volume and deep well 96-, 384and 1536-well plates that conform to propose ANSI standards
submitted by the Society for Biomolecular Sciences. In addition, the
TETRAcycler handles Molecular Devices qualified tips. Reservoirs can be
used during robotic integration, however the TETRAcycler is not able to
move these. All reservoirs must be loaded manually prior to running an
experiment, including the Cell Reservoir.
0112-0109 H 23
System Hardware Features
Note: While the system is compatible with plates that conform to
proposed ANSI standards submitted by the Society for Biomolecular
Sciences, some plates may not be handled as reliably by the
TETRAcycler due to their low plate weight. During robotic integration,
it is recommended that handling of the plates and tips by the
TETRAcycler be evaluated for plate handling robustness prior to
starting a screen.
The FLIPR® Tetra System’s upper and lower door must remain closed
for the duration of experiment. Plates are transported in and out of the
instrument only by the TETRAcycler system’s robotic landing pad door.
WARNING! Do not place your fingers in the TETRAcycler
shuttle door as this may cause injury.
SynchroMax ET™
The SynchroMax ET is a six-stack plate handler available as an optional
purchase with the FLIPR® Tetra System. It delivers plates to and from
the landing pad, integrating with the TETRAcycler, which ferries the
plates to and from their appropriate locations in the five-position stage.
The configuration interface of the SynchroMax ET software is opened
directly from within ScreenWorks Software, making experiment
configuration straightforward.
Observation Panel
In order to view hardware movements in the upper top compartment
while troubleshooting the FLIPR® Tetra System, use the observation
panel. Under normal operating conditions the upper door must be
closed in order to run an experiment, ensuring no light enters the
chamber. When the observation panel is mounted to the chamber,
however, the door can be left open, allowing you to view movements of
the pipettor and TETRAcycler. Normal instrument control is performed
via ScreenWorks Software, SynchroMax ET, or third-party plate
handling software. For the Aequorin ICCD camera test images are
displayed.
The observation panel is stored attached to the inside of the upper
door. To mount the panel, remove it from the door and attach it with
the four captive thumbscrews to the top compartment frame.
24 0112-0109 H
FLIPR® Tetra High Throughput Cellular Screening System User Guide
To acquire quality data, reaffix the observation panel to the inner door
prior to running an experiment.
WARNING! If pretending to run in luminescence mode with the
Aequorin ICCD camera, DO NOT touch the white door switches.
Room light will damage the Intensifier. The door switches
detect the open door to protect the camera.
Note: The observation panel should only be used to view internal
pipettor movement; it should not be used during experiments when
data is being accumulated. Test data (in the case of the Aequorin ICCD
camera) or compromised data (with the Standard camera) will be
shown if not collected under dark conditions with closed doors.
Liquid-Handling System
Compounds are transferred from source plates or reservoir to read
plates by the pipettor mounted on the rear wall of the top
compartment, above the TETRAcycler. The pipettor assembly can be
fitted with a standard pipettor head, to use disposable tips, or a pin tool
head, which uses solid or slotted pins to carry compound.
All 1536, 384, or 96 tips or pins operate at the same time,
simultaneously picking up compound from all the wells in a source plate
(or a quarter of the wells; see Compatible Plate Configurations on
page 30 below) or Cell Reservoir, and similarly dispensing these
simultaneously to the read plate. Fluid mixing steps can be configured
for source plates before compound is picked up, and for read plates
once it has been dispensed.
Pipettor heads are user-installable and can be interchanged in
approximately less than 5 minutes; see Exchanging Pipettor and Pin
Tool Heads on page 129.
Standard Pipettor Head
Standard pipettor heads are available in 1536-, 384- and 96-tip
formats.
The 384- and 96-pipettor heads both use disposable plastic tips. In
contrast, the 1536-pipettor head uses a stainless steel tip block with a
disposable 1536-tip gasket.
Plastic tips can be washed or replaced between each compound
addition or at the end of an experiment. The 1536-tip block is washed
at specified times.
0112-0109 H 25
System Hardware Features
Pipettor operations are controlled from within ScreenWorks Software
protocols, or some operations, for example, loading tips, can be
performed individually, directly through commands in the Instrument
menu. Connectors on the back of the pipettor head identify the head
format as 1536, 384, or 96 tips, so ScreenWorks Software only offers
valid plate formats and pipetting parameters for protocol setup.
The standard pipettor head uses air displacement to control aspiration
and dispense speed and volume. The volume of compound to be
transferred is configured in the software, and it is possible to draw
compound from multiple source plates to dispense into one destination
plate, or to aspirate from one plate and dispense to multiple well plates
or quadrants.
The 96- and 384-pipettor heads displace air in the disposable pipette
tips. In the 1536-pipettor head a plunger for each of the 1536 tips
presses against an elastic gasket seated on the tip block. When the
plungers move down they create an initial seal between the gasket and
tip block. Once the seal is created, further plunger movement causes
air displacement in the tip block (see Figure 2-4).
Figure 2-4 Seal creation in the 1536-pipettor head.
Minimum pipettor precision is as follows:
• 3% for 75 μL additions (96-well).
• 4% for 25 μL additions (384-well)
• 6% for 3 μL additions (1536-well)
Performance is dependent on tip/gasket seating and can be
compromised if the seal is broken. Use only Molecular Devices
recommended tips and gaskets to ensure the highest accuracy and to
reduce the possibility of damaging the pipettor. See Appendix C:
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FLIPR® Tetra High Throughput Cellular Screening System User Guide
Consumables and Accessories on page 283 for recommended tips.
Cell Suspension
The Cell Suspension option consists of two components:
• The Cell Reservoir installable in Position 4 (Source 3) in the 5
position stage.
• The Cell Suspension module located externally on the right side
of the instrument.
The Cell Reservoir (see Figure 2-5) is user installable. The Cell
Suspension module (see Figure 2-6) consists of a shelf with a magnetic
motor mounted underneath it, a cell flask with a magnetic stirrer, up to
four fluid bottles for automated cleaning, and a removable cover for
keeping cells in a dark environment.
Figure 2-7 demonstrates how the system is connected and shows all
the possible combinations for protocol development.
Figure 2-5 Cell Reservoir.
The Cell Reservoir, which the user places in a source plate location, is
filled from any of the bottles in the external Cell Suspension module by
a pump with adjustable speed and direction.
The Cell Reservoir is a special plate type that has one fluid line used for
both input and output, and an electronic plate ID that is part of the
electrical/fluidic connector which identifies the reservoir to the system.
In the case of an overflow, an overflow trough catches excess fluid and
pipes it to the tip washer overflow trough, which directs it to the
overflow bottle. The reservoir is also autoclavable for cleaning
purposes.
The spinner flask contains a stirrer, which is driven by a magnetically
coupled motor mounted in the lower part of the Cell Suspension
module. The Cell Suspension stir speed can be set in a protocol or with
manual instrument controls. Stir speed of 5 equals approximately 1
0112-0109 H 27
System Hardware Features
revolution per second. From the uniform suspension in the spinner flask
cells are pumped into the Cell Reservoir, where the pipettor head in 96,
384, or 1536 format removes the appropriate amount and cells are
automatically pumped to a specified destination. A protocol in
ScreenWorks Software controls the stir speed and source/destination of
cell suspension activity.
The cell valve selects the source for filling, or the destination for
draining. There are 8 valve positions: flask fill (for filling the reservoir),
flask drain (for draining the reservoir without causing air bubbles),
Waste Bottle A, Waste Bottle B, and Fluid 1–4.
Fluid 1–4 are user specifiable, and can be cleaning solutions, water or
buffer. The user can choose to pump cells back into the cell flask or to
any other fluid bottles. Bottles for Fluid 1 and 2 are automatically
included with the Cell Suspension option.
Cell Reservoir can be washed by either adding the Wash Reservoir
process to the protocol (see Constructing Protocols Using FLIPR Tetra
Processes on page 70), selecting Wash Reservoir in the Instrument
> Manual Operation menu, or manually removing the reservoir and
autoclaving it.
Figure 2-6 Cell Suspension Module.
28 0112-0109 H
FLIPR® Tetra High Throughput Cellular Screening System User Guide
Figure 2-7 Cell Suspension Module connections.
Pin Tool Head
Pin tools are blocks of solid or slotted pins, where the pins replace the
hollow tips used with a standard pipettor. The pins use capillary action
to pick up and transfer liquid from one plate to another. Their ability to
accurately and reliably transfer compounds in nanoliter volumes allows
users to supply test compounds in 100% DMSO solution, removing the
need to prepare intermediate dilution plates.
The volume that each pin picks up is determined by the size of the pin
(and, if slotted, the size of the slot) and the withdrawal speed of the pin
from the liquid—a faster removal speed leaves more liquid on the pin.
Pins for the 384 pin tool are supplied in four sizes, giving a total range
(across all these sizes) from 84 nL to 656 nL. The 1536 pin tool has
seven different pin sizes, giving a total range from 19 nL to 117 nL.
0112-0109 H 29
System Hardware Features
Each pin size has a specified volume range that it carries:
• The lowest reported volume is for a tip removal speed of 7.8
• The highest reported volume is for a tip removal speed of 57.0
The precise volumes that will be picked up at given tip removal speeds
should be determined by users in assay development.
The FLIPR® Tetra System can be fitted with 384- or 1536-pin tool
heads. Pin tools themselves, in the appropriate 384 or 1536 format,
can be easily and rapidly replaced to change the pin size.
All the pin tools used with the FLIPR® Tetra System are available with a
hydrophobic and lipophobic coating to prevent or reduce the
nonspecific binding of proteins and lipids to the pins.
In order to ensure uniform compound pick-up across the entire pin tool,
pins can be configured to ‘float’ in source plate wells. Individual pins
are not rigidly attached to the pin block, having a small amount of
vertical movement up into the block. When set to float, the pin head
moves down very low so that all pins sit on the bottom of the well and
push up a little into the block. This ensures that all pins are equally
immersed in their wells, i.e., sitting on the bottom. This outcome could
not be guaranteed if the pins were rigidly fixed to the block, given that
plate bottoms are often not completely flat.
mm/s.
mm/s.
Compatible Plate Configurations
The 96- and 384-pipettor heads can be used with source or read plates
with equal or one order higher well number. This is because the FLIPR®
Tetra System can aspirate or dispense into quadrants of a plate. The
following combinations are possible:
• The 96-pipettor head can be used with 96- and 384-well plates.
• The 384-pipettor and pin tool head can be used with 384- and
1536-well plates.
• The 1536-pipettor head can be used only with 1536-well plates.
Deep-well plates or reservoirs can be substituted for standard well
plates.
When compound is aspirated or delivered to a plate with a greater
number of wells than the pipettor head, the quadrant number (1 to 4)
must be entered in the protocol configuration in ScreenWorks Software
for each dispense.
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