Each homogeneous assay kit utilizes a proprietary indicator dye and quencher combination to maximize cell line/channel/compound applicability, while eliminating causes of data variability. This unique formula responds 10 times faster and has greater temperature stability than traditional dyes, providing high quality screening data that shows good correlation with manual patch clamp assays.
Because ion channel activity is highly sensitive and potentially impacted by subtle chemical changes, two FLIPR® Membrane Potential Assay Kits (Red and Blue) are available to select the optimal conditions for your delicate ion channel targets. Both formulations utilize Molecular Devices proprietary quench technology to enhance signal windows and yield acceptable Z-scores to screen a variety of targets, including TRP, ligand-, cyclic nucleotide- and voltage-gated channels.
Unlike traditional dyes such as DiBAC, FLIPR Membrane Potential Assay Kit detects bidirectional gradient changes so you can monitor both variable and control conditions within a single experiment. We recommend evaluating both assay kits to discern which formulation is right for your target.
Comparison of Patch Clamp and FLIPR® Membrane Potential Assay Kit Data
The manual patch clamp method is able to detect fast responses allowing detection of very rapid changes in membrane potential. Comparing data generated using the FLIPR Membrane Potential Assay Kits with results from the manual patch clamp method shows good correlation (Figures 1 and 2). Both the opening and closing of the ion channel can be observed. This differs from DiBAC, which can only show unidirectional changes in membrane potential (Figure 3).
Figure 1: Comparison between patch clamp (mV) and FLIPR Membrane Potential (fluorescence) Assays on CHO cells expressing a voltage-gated K+ channel*.
Figure 2: Correlation of changes in membrane potential to fluorescence assays on the FLIPR Instrument: CHO cells transfected with K+ channel exposed to various concentrations of potassium*.
Figure 3: Comparison between FLIPR Membrane Potential Assay Kit, DiBAC, and Fluo-3 assays on ligand-gated Ca2+ channels*.
*Data courtesy of Michael Xie, Millenium Pharmaceuticals.
Two Membrane Potential Kit Quench formulations are available
Because ion channel activity is sensitive to interference, and chemical interference with a particular ion channel is highly unpredictable, the FLIPR Membrane Potential Assay Kits have two formulations. Both formulations combine the advantages of Molecular Devices proprietary membrane potential indicator dye with our patented quench technology. One formulation uses a blue quencher and the other formulation uses a red quencher. We recommend that both versions be evaluated for each individual target/cell line to determine which formulation will provide optimal performance.
Figure 4: Comparison between FLIPR Membrane Potential Assay Kit, DiBAC,
and Fluo-3 assays on ligand-gated Ca2+ channels*.
Fluorescence intensity changes with increase or decrease in cellular membrane potential
FLIPR Membrane Potential Assay Kits detect ion channel modulation by increasing or decreasing the fluorescent signal as cellular membrane potential changes. The fluorescent signal increases in intensity during membrane depolarization as dye follows the positively charged ions inside the cell. During membrane hyperpolarization, fluorescent signal decreases in intensity as dye follows the positively charged ions out of the cell. The kits are uniquely suited for use with the simultaneous pipet and read capability of the FLIPR® Tetra System and FlexStation® 3 Microplate Readers to capture fast kinetics associated with ion channel activation.
The kits employ a quenching dye to reduce background fluorescence and improve the signal-to-noise ratio. The patented quench technology (U.S. patent number 6,420,183, EPO patent number 0 906 572) is offered to drug discovery and life science researchers exclusively by Molecular Devices through the purchase of FLIPR Assay Kits.