CHEMBL3706248

Inhibition Assay: Inhibition of CRAC channels was determined following thapsigargin (Sigma, Cat # T9033) induced endoplasmic calcium release in Jurkat cells. (see Yasurio Yonetoky et. al Bio. & Med. Chem. 14 (2006) 4750-4760). Cells were centrifuged and resuspended in equal volumes f Ca2+ and Mg2+ free Hanks buffer and Fluo-8 NW dye (ABD Bioquest, Inc., Sunnyvale, Calif.) loading solution at 2x105 cells/100 ul/well in 96-well black plate. Plate is incubated at 37 C./5% CO2 for 30 mM followed by further 15 mM incubation at room temperature. Test compounds (DMSO stocks diluted in Ca2+ and Mg2+ free Hanks buffer) at desired concentrations were added to the wells and incubated for 15 mM. Thapsigargin (1 uM final concentration) was added to the wells and incubated for 15 min to inhibit the Sarco-endoplasmic reticulum Ca2+ ATPase pump thereby depleting endoplasmic calcium and raising cytosolic calcium concentrations. Store-operated calcium entry was initiated by adding extracellular Ca2+.