CHEMBL3706069

Calcium Mobilization Assay: The cDNA for rat metabotropic glutamate receptor 5 (rmGluR5) and the cDNA for human metabotropic glutamate receptor 5 (rmGluR5) were generous gifts from S. Nakanishi (Kyoto University, Kyoto, Japan). The rmGluR5 or rmGluR5 was stably expressed in a HEK 293 cell line and grown in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, Calif.) with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin, 100 ug/mL streptomycin and 0.75 mM G1418) at 37 C., 5% CO2. Twenty-four hours prior to assay, cells were seeded into 384-well black wall microtiter plates coated with poly-D-lysine. Just prior to assay, media was aspirated and cells dye-loaded (25 uL/well) with 3 uM Fluo-4/0.01% pluronic acid in assay buffer (Hank's Balanced Saline Solution (HBSS)): 150 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, plus 20 mM N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, 0.1% bovine serum albumin (BSA) and 2.5 mM probenicid).