| Assay Method Information | |
| | LPAR1 Calcium Flux Assays |
| Description: | A cDNA encoding the human LPAR1 receptor is synthesized and cloned into pDNA3 expression plasmid. The plasmid is transfected in U937 cells using Lipofectamine 2000 (Invitrogen Corp., USA). Clones stably expressing human LPAR1 are selected using puromycin and identified as cells that show Ca-influx in response to LPA.U937 cells overexpressing human LPAR1 are seeded at 100,000 cells per well in a 96-well fibronectin (10 ug/ml) coated plate in 60 μl of assay buffer (HBSS containing 20 mM HEPES and 0.2% BSA) and then incubated for 60 minutes. Then 50 μl of a calcium indicator dye (Fluo-4 NW, Molecular Probes) are added to each well and incubation continued for 30 minutes at 37° C. and then 30 minutes at room temperature. 50 μl of test compounds in 4% DMSO are added to the cells and incubation continued at room temperature for 40 minutes. Cells are the stimulated by the addition of 50 μl of 128 nM LPA and intracellular calcium is measured using the FLIPR TETRA (Molecular Devices). IC50 values are determined using Genedata Screener analysis tool. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |