Lance ultra camp kit

Homogeneous time-resolved fluorescence energy transfer (HTRF) assays were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA), based on competitive displacement of a europium chelate-labelled cAMP tracer bound to a specific antibody conjugated to acceptor beads. The optimal cell density for an appropriate fluorescent signal was first established by measuring the TR-FRET signal determined as a function of forskolin concentration using different cell densities. Forskolin dose-response curves were related to the cAMP standard curve in order to establish a cell density with a response covering most of the dynamic range of the cAMP standard curve. Cells were not treated or treated with vehicle or 4 μM of the indicated TM peptides for 4 h at 37 °C in an atmosphere of 5% CO₂. Cells were then grown (800 cells/well) in white ProxiPlate 384-well microplates (PerkinElmer, Waltham, MA) in medium containing 50 μM zardaverine, stimulated with agonists for 10 min before adding 0.5 μM forskolin or vehicle and incubated for an additional 15-min period. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).

Experiments were performed using the LANCE Ultra cAMP kit (Perkin Elmer) with cells in suspension according to the manufacturer’s instructions with minor modifications. Briefly, cells were harvested using versene (Gibco, Thermo Scientific), spun at 100×g for 5 minutes and resuspended in assay buffer (HBSS, 5 mM HEPES, 0.5 mM IBMX (Sigma-Aldrich), 0.1% BSA, pH 7.4) at 8,000 cells/well for hNPSR-107N, 4,000 cells/well for hNPSR-107I, and 20,000 cells/well for mNPSR. Serial dilutions of test compounds and NPS were prepared at 2× the desired final concentration in assay buffer. Cells were transferred to 96-well ½ area white polystyrene plates, and NPS or test compounds were added at the indicated concentrations. Plates were incubated for 30 minutes (hNPSR-107N or hNPSR-107I) or 1 hour (mNPSR) at room temperature. After addition of Eu-tracer cAMP and ULight-anti-cAMP, plates were incubated for 1 hour at room temperature in the dark. Data was collected on a FlexStation III instrument (excitation at 340 nm, emission at 615 and 665 nm, Molecular Devices) or a CLARIOstar (excitation at 340 nm, emission at 620 and 665 nm, BMG LABTECH). TR-FRET data in relative fluorescent units (RFU) was converted to fmol cAMP through interpolation using a cAMP standard curve and data were fit using a three-parameter non-linear regression to generate EC₅₀ values (GraphPad Prism, 6.0).