| Assay Method Information | |
| | Beta-Arrestin Recruitment Assay |
| Description: | The Tango™ EDG2-bla U2OS cells are obtained from Invitrogen. These cells contain the human LPA1 receptor cDNA linked to a TEV protease site and a Gal4-VP16 transcription factor integrated into the Tango™ GPCR-bla U2OS parental cell line. This parental cell line stably expresses a beta-arrestin/TEV protease fusion protein and the beta-lactamase (bla) reporter gene under the control of a UAS response element. Upon LPA (agonist) binding, LPA1 receptor gets activated, leading to arrestin-protease recruitment and proteolytic release of the transcription factor: The transcription factor then regulates transcription of a beta-lactamase reporter construct, which is measured upon addition of the live-cell substrate.10′000 Tango™ EDG2-bla U2OS cells are seeded in a 384-well black with clear bottom plate in 30 μl Freestyle 293 Expression Medium (Invitrogen) and incubated for 20 h at 37° C., 5% CO2. For antagonist assays, 5 μl of test compound (dilution series in DMSO/Freestyle 293 Expression medium/0.1% fatty acid free BSA (Sigma) or buffer control are added per well and incubated for 30 min at 37° C., 5% CO2. 5 μl of LPA 18:1 (500 nM final) (solution in Freestyle 293 Expression medium/0.1% fatty acid free BSA (Sigma)) are added per well and the plate incubated for 16 h at 37° C., 5% CO2. Cells are then loaded with LiveBLAzer-FRET™ B/G Substrate (Invitrogen) for 2 h in the dark and the fluorescence emission at 460 nm and 530 nm is measured using the SynergyMx reader (BioTek). Following the background subtraction from both channels, the 460/530 nm emission ratio for each well is calculated, then plotted and fitted to a 4-parameter logistic function to obtain IC50 values. IC50 is the concentration of antagonist inhibiting 50% of the maximal response. |
| Affinity data for this assay | |
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