Antagonist activity at human LPA5 receptor expressed in rat B103 cells assessed as inhibition of LPA-induced calcium mobilization preincubated for 30 mins followed by LPA induction by FLIPR Calcium 4 dye-based fluorometric analysis
Receptor Assay: Engagement of the LPA5 receptor by its ligand, oleoyl-L-α-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA5 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Minimal Essential Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom poly-D-lysine coated 384 well plate (Corning). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1x Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake.
Agonist activity at FLAG-tagged LPA5 receptor (unknown origin) expressed in rat B103 cells assessed as increase in intracellular calcium level up to 10 uM measured every 3.42 secs for 70 secs by Fura-2-AM dye based fluorescence assay
Antagonist activity at FLAG-tagged LPA5 receptor (unknown origin) expressed in rat B103 cells assessed as inhibition of LPA-induced intracellular calcium level up to 100 uM measured every 3.42 secs for 70 secs by Fura-2-AM dye based fluorescence assay
Agonist activity at FLAG-tagged LPA5 receptor (unknown origin) expressed in rat B103 cells assessed as increase in intracellular calcium level measured every 3.42 secs for 70 secs by Fura-2-AM dye based fluorescence assay