| Assay Method Information | |
| | Determination of the Inhibitory Activity of the Compounds on Human P2X3 and P2X2/3 Receptors |
| Description: | Cells were seeded into a poly-D-lysine-coated 384-well cell culture plate (Corning) at a density of 11,000 cells/well/25 μL of cell inoculation medium, and were cultured in a cell incubator overnight. On the day of the test, the calcium 6 dye was diluted to a 2× concentration with a assay buffer, 25 μL of the 2× calcium 6 dye was added to the 384-well cell culture plate, which was incubated at 37° C. for 2 hours, and then placed at room temperature for further use. The test compound and the agonist, α,β-MeATP were diluted to a 7× concentration with the assay buffer, 10 μL of the 7× test compound was added to the 384-well cell culture plate, which was incubated at room temperature for 15 minutes, and 10 μL of the 7× α,β-MeATP was transferred into the 384-well cell culture plate. The data were measured and analyzed using FLIPR Tetra, and the half inhibitory concentration (IC50) of the test compound on P2X3 and P2X2/3 receptors was calculated with the GraphPad Prism four-parameter equation. |
| Affinity data for this assay | |
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