Intracellular calcium mobilization assay

Intracellular Ca²⁺ release was measured as described.⁽²²⁾ In brief, HEK and HEK-CaSR cells were transfected with plasmids and seeded at 10,000 cells per well in black, clear-bottom 384-well plates. Forty-eight hours posttransfection, cells were starved in starvation media supplemented with 0.1 mM Ca²⁺ for 5 hours before the assay. Cells were loaded with FLIPR Calcium 5 Assay Kit dye (Molecular Devices) prepared in assay buffer (Hank’s balanced salt solution [HBSS] [without Ca²⁺ and Mg²⁺]+ 0.1 mM Ca²⁺ + 20 mM HEPES + 2.5 mM probenecid at pH 7.4) for 1 hour at 37°C. P-15, Ca²⁺, and DMSO (vehicle) were prepared at various concentrations in assay buffer. After 20 s baseline reading, compounds were dispensed on a cell plate and changes in fluorescence intensity were measured for another 100 s in a FLEXStation III plate reader (Molecular Devices). Data in HEK-CaSR cells were normalized to the maximum response (elicited by 7.49 mM Ca²⁺ + vehicle) in pcDNA3.1-transfected cells and were plotted as maximum-minimum signal at each concentration. Data from P-15–stimulated HEK cells were normalized to corresponding volumetric concentration of vehicle and were plotted as described for HEK-CaSR cells.