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Assay Method Information

Assay Name:  In Vitro LPA1 Receptor Assay
Description:  Engagement of the LPA1 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 LPA1 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 (Dulbecco's Modified Eagle Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom collagen coated 384 well plate (Beckton Dickinson). 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 (1 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 1 Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37 C. for one hour to allow for dye uptake. To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) were added to the assay wells and allowed to incubate for twenty five minutes. After incubation with test compounds the assay plate was placed in a FLIPR Tetra system (Molecular Devices) and LPA (diluted in assay buffer) was added to give a final concentration equivalent to its determined EC80 against LPA1 receptor. Ligand-dependent changes in intracellular calcium levels were determined by measuring changes in fluorescence of the dye over 515-575 nM following excitation over 470-495 nM.
Affinity data for this assay
 

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Last update November 1, 2007
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