| Assay Method Information | |
| | In Vitro Test of Inhibition of Human FP Receptor Activity (B-1B) |
| Description: | For the characterization of test substances in respect of FP antagonism, the PGF2α-induced calcium flux in recombinant FP-expressing CHO cells which additionally express the Ca2+ sensor protein GCaMP6 was used.3000 cells in 25 μl of full medium [DMEM F12, 10% FCS, 1.35 mM sodium pyruvate, 20 mM HEPES, 4 mM GlutaMAX, 2% sodium bicarbonate, 1% Pen/Strep, 1% 100× non-essential amino acids] are sown per well of a 384 multititer plate (from Greiner, TC plate, black with clear base) and incubated at 37° C., 5% CO2 for 24 hours. Prior to the measurement, the medium is replaced by 30 μl of buffer [calcium-free Tyrode (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl2, 4.8 mM NaHCO3, pH 7.4), 2 mM CaCl2, 0.01% BSA] and incubated at 37° C., 5% CO2 for 30 minutes. The test substance is prepared in DMSO in various concentrations as a dose-response curve (starting concentration 10 mM, dilution factor 3.16) and prediluted 1:50 with calcium-free Tyrode/2 mM CaCl2/0.01% BSA. 10 μl of the prediluted substance solution are added to the cells and incubated at 37° C., 5% CO2 for 10 minutes. The FP receptor is activated by adding 20 μl of 3 nM (final concentration) PGF2α in calcium-free Tyrode/2 mM CaCl2, and the calcium flux is determined by measuring the fluorescence at ex. 470 nm/em. 525 nm in a fluorescence measuring instrument (FLIPR Tetra, MolecularDevices) for 120 seconds. |
| Affinity data for this assay | |
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