CHEMBL3706169

FLIPR Calcium Assay: Antagonizing effect of compounds for human GnRHR1 was evaluated by change of calcium levels in GnRH-stimulated cells. After removing the culture medium of HEK293 cells transiently expressing human GnRHR1, cells were washed with 200 .mu.L piper well of the washing buffer (Hank's Balanced Salt Solutions, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1.3 mM calcium chloride, 0.5 mM magnesium chloride, 0.4 mM magnesium sulfate). One hundred .mu.L of the Ca.sup.2+sensitive dye solution (FLIPR Calcium Assay Kit (Molecular Devices)) was added to the well, and the cells were incubated for 1 hour at 37.degree. C., 5% CO.sub.2. Then, intracellular calcium levels were determined under the following condition by using FLEX STATION (Molecular Devices). In the equipment, which was warmed to 37.degree. C., 50 .mu.L of test compound diluted with the measurement buffer (the washing buffer with 0.1% Albumin bovine serum) was added to the well.