Htrf camp hirange kit

The HTRF cAMP assay was performed as previously published with modifications [17 (link)]. Cellular cAMP levels were measured using reagents supplied by Cisbio International (HTRF cAMP HiRange kit). Cultured cells were washed twice with phosphate-buffered saline (8.1 mM NaH₂PO₄, 1.5 mM KH₂PO₄, 138 mM NaCl, and 2.7 mM KCl, pH 7.2), and then dissociated in phosphate-buffered saline containing 1 mM EDTA. Dissociated cells were collected by centrifugation for 5 min at 2000g. The cells were resuspended in cell buffer (DMEM plus 0.2% fatty acid free bovine serum albumin) and centrifuged a second time at 2000g for 5 min at 4 °C. Subsequently, the cells were resuspended in an appropriate final volume of cell buffer plus the phosphodiesterase inhibitor Ro 20–1724 (2 μM). 5000 cells were added at 5 μl per well into 384-well, round bottom, low volume white plates (Grenier Bio One, Monroe, NC). Compounds were diluted in drug buffer (DMEM plus 2.5% fatty acid free bovine serum albumin) and added to the assay plate at 5 μl per well. Cells were treated with drugs or vehicle for 1 h in a humidified incubator at 37 °C and 5% CO₂. d2-conjugated cAMP and Europium cryptate-conjugated anti-cAMP antibody were added to the assay plate at 5 βl per well. After 1-h incubation at room temperature, the plate was read on a TECAN GENious Pro microplate reader.

The HEK293T cells (ATCC, Manassas, VA) were transiently transfected with wild-type and mutant RXFP1 expression constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were tested in the cAMP assay 48 hr after transfection. Cellular cAMP level was measured using HTRF cAMP HiRange kit (CisBio, Bedford, MA, USA) according to manifacturer’s protocol. Cells were stimulated with various concentrations of porcine relaxin peptide (RLN) or ML290. Porcine relaxin peptide was a gift from Dr. O. David Sherwood (University of Illinois at Urbana-Champaign).^{25 (link)} The signal was read on a FLUOstar Omega (BMG Labtech, Cary, NC) plate reader. All experiments were repeated at least three times in triplicates. Ligand activity is reported through two measurements: EC₅₀ (concentration necessary to reach 50% of the maximum cAMP signal produced by the molecule) and maximum response (E_max, corresponding to the level of cAMP elevation normalized to FSK control). Statistical processing of the data was performed using GraphPad Prism software (San Diego, CA).

Cells were lifted with non-enzymatic cell stripper and resuspended in assay buffer at desired concentrations. cAMP assays were performed according to the manufacturer’s protocol using CisBio’s HTRF cAMP HiRange Kit. Cells were incubated with compounds for 30 minutes at 37°C. The reaction was terminated by sequentially adding D2-labeled cAMP and cryptate-labeled anti-cAMP antibody in lysis buffer. The plate was incubated at room temperature for 60 minutes before reading fluorescent emissions at 620 nm and 668 nm with excitation at 314 nm on FlexStation III (Molecular Devices). Cyclic AMP assay results are shown as “Ratio 668/620 × 10,000” (ratio of fluorescence at 668 nm and 620 nm x 10,000). Data in graphs are represented in Mean ± SD. Dose-dependent responses were fitted with sigmoidal dose-response curves allowing variable slopes using GraphPad Prism version 6. The graphs display dose-dependent stimulation of intracellular cAMP level upon treatment with control ligand or compounds.