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| | Rubidium efflux assay |
| Description: | HEK-293 cells overexpressing the channel of interest were seeded in a 96-well plate at a density of 30000 cells/well and incubated at 37° C./5% CO2 for 48 hr. The assay was carried out using the Red Membrane Potential Dye (Molecular Devices) following the manufacturer's instructions. Briefly, the cells were incubated with 1× red membrane potential dye for 1.5 hour. The cells were then treated with various concentrations of the test compounds for 15-20 min followed by depolarization with 10-30 μM Veratridine. The fluorescence was read following excitation at 510-545 nm and emission at 565-625 nm in FLIPR. The max-min fluorescence values were used to calculate the % inhibition. IC50 values were calculated by plotting % inhibition against concentration and curve fitting into a sigmoidal dose response.Through the use of the above described assay procedure, compounds were found to exhibit functional activity against Nav 1.7 in vitro and particularly well suited for the treatment of diseases or disorders as described herein above. |
| Affinity data for this assay | |
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