| Assay Method Information | |
| | FLIPR Calcium 4 Assay |
| Description: | The antagonistic activity of human P2X3 receptor (hP2X3) antagonists against hP2X3 was evaluated using the FLIPR Calcium 4 Assay Kit (Molecular Devices, R8141) and FLIPR TETRA instrument (Molecular Devices, 0296) to detect calcium flux signals. 24 h before the experiment, human cells stably transfected with the hP2X3 receptor were seeded into a 384-well plate at a density of 2×105 cells/mL, with 50 μL of cell suspension per well. The cells were then incubated in a 5% CO2 incubator at 37° C. for 16-24 h. Each test compound was prepared in DMSO at 180 times the desired concentration (20-50 mM DMSO stock solution). 500 nL of the solution was then added to each well of the 384-well plate, followed by addition of 30 μL of FLIPR Assay buffer (lx HBSS containing 1.26 mM Ca2++2 mM CaCl2), 20 mM HEPES). The mixture was shaken for 20-40 min to ensure homogeneous mixing. An agonist (α,β-meATP) was prepared using FLIPR Assay buffer at 3 times the desired concentration (desired final concentration of 400 nM). 45 μL of the agonist was then added to each well of another 384-well plate. The cell culture plate prepared one day ago was taken, and the cell supernatant was aspirated and discarded. 30 μL of Dye (FLIPR® Calcium 4 Assay Kit, diluted in FLIPR buffer) was added to each well. The plate was then incubated for 1 h. 15 μL of the compound was added to the cells in each well (using the FLIPR instrument). After 15 min, 22.5 μL of the agonist was added to each well. The fluorescence signal was detected (with an excitation wavelength of 470-495 nm and an emission wavelength of 515-575 nm). Taking the difference between the peak and trough values of the signal as the base data, the data for the highest concentration of the positive drug as the 100% inhibition rate, and the DMSO data as the 0% inhibition rate, the inhibition effect curve of the compound was fitted on the software Graphpad Prism 6, and the IC50 value was calculated. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |