Lance ultra camp detection kit

Inhibition of forskolin-stimulated cAMP production was measured using a LANCE Ultra cAMP Detection kit (PerkinElmer) following the manufacturer’s instructions. Flag-tagged wild-type CCR5 and mutants were cloned into the expression vector pTT5 (Invitrogen) and expressed in HEK293F cells (Invitrogen). Cells were harvested 48 h posttransfection. The cell-surface expression of CCR5 was measured as above described. The harvested cells were plated in 384-well plates (1,000 cells per well) using HBSS buffer (Invitrogen) supplemented with 5 mM HEPES (pH 7.5), 0.1% BSA (Sigma), and 0.5 mM IBMX (Sigma). Cells were stimulated with 2 μM forskolin (Sigma) and different concentrations of MIP-1α or RANTES (0.1 pM–1 μM diluted in stimulation buffer; see below for expression and purification of the chemokines) for 30 min at room temperature. Plates were then treated with the Eu-cAMP tracer and ULight-anti-cAMP working solution for 60 min at room temperature. Fluorescent measurements were acquired by an EnVision multilabel plate reader (PerkinElmer) with excitation at 330 nm and emission at 665 nm. The values were then converted to cAMP production by a standard dose-response curve. EC₅₀ and pEC₅₀ ± SEM were calculated using GraphPad Prism 8.0.

To measure intracellular cAMP concentrations, H9c2 cells and NMCMs were pretreated with 3-isobutyl-1-methylxanthine (0.1 mM, IBMX, Sigma-Aldrich) for 30 min and then stimulated with the non-selective MC-R agonist α-MSH (0.1 nM) or the MC5-R agonist PG-901 (0.1 nM) for 5, 15, 30, and 60 min. Cells were thereafter lysed with 0.1 M HCl and assayed for cAMP levels with a commercial kit (Cyclic AMP Select ELISA kit, Cayman Chemical, #501040) according to manufacturer’s instructions. Results were normalized against total protein concentrations (Pierce™ BCA Protein Assay Kit, ThermoFisher) and expressed as percentage of control samples that were left untreated.
To study concentration responsiveness of the compounds and Gα_i-evoked inhibition of intracellular cAMP production, a different method using LANCE Ultra^® cAMP Detection Kit (PerkinElmer, # TRF0262) was employed. For this purpose, cells were harvested, pipetted into a 96-well OptiPlate (6000 cells/well) and stimulated with different concentrations of α-MSH or PG-901 for 30 min. cAMP levels were determined based on changes in time-resolved fluorescence resonance energy transfer (TR-FRET) signal according to the manufacturer’s instructions. In order to detect agonist-induced reduction in cAMP levels, cells were treated with α-MSH or PG-901 in the presence of forskolin (1 μM) or isoprenaline (10 μM).