Table 2RXFP1 cAMP confirmatory assay protocol

SequenceParameterValueDescription
1Cells30 μL8,000 cells/well
2Time16–24 hIncubate at 37 °C and 5% CO2
3Reagent2 μL1,600 μM Ro 20–1724 in PBS
4Reagent2.5 μLCompounds in DMSO, forskolin as control
5Time30 minIncubate at 37 °C and 5% CO2
6Reagent8 μLcAMP-d2 diluted 1:18 in lysis buffer
7Reagent8 μLK-α-cAMP diluted 1:20 in lysis buffer
8Time30 minRoom temperature incubation
9DetectorTR-FRETEnVision plate reader

From: Discovery, optimization, and biological activity of the first potent and selective small-molecule agonist series of human relaxin receptor 1 (RXFP1)

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