Inhibition of forskolin stimulated cAMP accumulation assays

CHO-OPRD1 cells were grown to confluence then harvested and resuspended at 1e⁶ cells per ml in assay buffer (HBSS+25 mM HEPES,+0.05% BSA). Increasing concentrations of BMS-986187 (30 nl of 100 × final concentration in 100% DMSO) were added to separate rows of 1,536-well white solid NT plates by acoustic dispense using an Echo-550 (Labcyte, CA). Next, 1 μl of increasing concentrations of naloxone (at 3 × final concentration in assay buffer) were added to separate columns of the plates. Next, 1 μl of cells (1,000 cells per well) were added to all wells followed by 1 μl of forskolin (3 × final concentration in assay buffer). Plates were lidded and incubated for 45 min at RT. Incubations were terminated by the addition of Lance-Ultra cAMP detection reagent (1.5 μl of Eu-cryptate-labelled cAMP tracer in lysis buffer, followed by 1.5 μl of U-light-conjugated anti-cAMP antibody in lysis buffer). After a 1 h incubation at RT, time-resolved fluorescence was detected on a Viewlux or Envision plate reader (PerkinElmer) with excitation at 337 nm and emission reads at 615 and 665 nm. The ratiometric data (665 nm read/615 nm read) × 10,000 were then converted to cAMP (nM) based on a standard curve for cAMP (replacing the cell addition step) run at the same time and under identical conditions to the assay. Lance-Ultra cAMP detection reagents were from PerkinElmer Life Sciences. All other chemicals, unless otherwise specified, were from Sigma.