Assay Method Information
Assay Name:  Evaluation of In Vitro Biological Activity
Description:  The antagonist property of the compounds disclosed herein was determined using the FLIPR (fluorescence imaging plate reader) method, showing that the compounds are inhibitors for the intracellular calcium increase induced by the activation of hP2X3 (human purinergic P2X receptor subtype 3, Accession No. NM_002559.4) expressed in HEK293 cells (human renal epithelial cell line, ATCC). HEK293 cells that stably express hP2X3 were cultured in DMEM high glucose medium containing 10% FBS (fetal bovine serum, Gibco, 10099-141), 1% penicillin-streptomycin (Gibco, 15140-122) and 1 mg/mL G418 (Invitrogen, 10131027) in a cell incubator at 37 C., 5% humidity. Cells at 400,000 cells/mL were seeded into a 384-well plate (10,000 cells/well) 18-24 h prior to the FLIPR experiment and then incubated overnight in a cell incubator. On the day of the experiment, the medium was discarded and the cells were washed in an FLIPR buffer (each 30 mL buffer contains 0.3 mL of probenecid (Thermo, P36400), 0.6 mL of 1 M HEPES (Invitrogen, 15630080) and 29.1 mL of HBSS (Invitrogen, 14065056)). Each well was added with 20 μL of 0.5 Calcium 6 fluorescent dye (Molecular Devices, R8190) and then was subjected to dye-loading incubation at 37 C. for 1.5 h. Each well was added with 10 μL of test compound (which was dissolved in DMSO at a concentration of 10 mM and serially diluted with buffer) or vehicle, and then was left to equilibrate for 30 min at room temperature. The cell plate was then placed in the FLIPR for baseline fluorescence measurements (excitation at 485 nm and emission at 525-535 nm). An agonist (BZ-ATP (Sigma, B6396) at a final concentration of 2.5 μM) or a vehicle (ultrapure water) was then added at 10 μL/well, fluorescence values were measured for 2 min at 1-second intervals, and finally the output fluorescence counts were analyzed.
Affinity data for this assay
 
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