Assay Method Information | |
| Intracellular Calcium Mobilization Assay |
Description: | MRGPRX2 stable cells were maintained in DMEM containing 10% FBS, 100 μg/ml hygromycin B, and 15 μg/ml blasticidin. For the calcium mobilization assay, cells were plated into glass bottomed, poly-L-lysine coated, black 384 well plates at a density of 20,000 cells/well in medium containing 1% dialyzed FBS, 1 μg/ml tetracycline, 100 IU/ml penicillin and 100 μg/ml streptomycin and incubated 24 h. Following tetracycline induction, medium was removed and cells were loaded with 20 μl/well of 1× FLIPR Calcium dye (Molecular Devices) and 2.5 mM probenic acid, for 1 h in a humidified environment at 37 °C with 5% CO2. For mast cell calcium experiments, cells were seeded at a density of 50,000 cells/well in Tyrode's buffer containing 1× calcium dye and incubated for 1 h before treatment and analysis. After dye loading, baseline was measured for 10 s before drug treatment, and then cells were treated with 10 μl of 3× concentrated drug in drug buffer (1× HBSS with 20 mM HEPES and 2.5 mM probenic acid, pH 7.4) in 16 point concentration response curves from 100 μM to 0.003 nM. |
Affinity data for this assay | |
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