Ca2+ Fluorometric Imaging Plate Reader (FLIPR) Assays
Cells were plated at 10,000 cells per well in 384-well tissue culture treated black walled clear bottom assay plates (greiner #781091) and maintained overnight at 37°C with 5% CO2. After removal of the growth media, cells were incubated with 20µl Ca2+-6 dye in assay buffer (HBSS with 20mM HEPES, 1mg.ml-1 glucose and 0.01% pluronic) containing 2.5mM probenecid for 60 minutes at 37°C and 5% CO2. Antagonists and β-alanine were prepared in assay buffer. Antagonists were pre-incubated with the cells and dye for 30 minutes prior to addition of agonist and changes in intracellular Ca2+were monitored kinetically over a 6-minute period using the FLIPR; five baseline measurements were taken, prior to addition of agonist, followed by 60 measurements at 1 second intervals and by 60 measurements at 5 second intervals.