Alphascreen camp assay kit

After transfection (72 h), cells were washed once with DMEM/F12 containing 1 mM 3-isobutyl-methyl-xanthine (IBMX) followed by incubation in the presence of the indicated compounds and forskolin (2.5 µM) for 15 min at 37 °C. The final concentration of DMSO was 1%. Cells were lysed in 25 μl lysis buffer (5 mM HEPES; 0.1% BSA; 0.3% Tween20; 1 mM IBMX; pH 7.4) and kept frozen at −20 °C until measurement. To measure cAMP concentration, the AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) was used according to the manufacturer’s protocol.

U2Os cells expressing the P2Y₁₂ receptor were cultured in DMEM medium containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 2 µmol/ml glutamine. For the assay of cAMP accumulation, cells were plated in 96-well plates in 100 µl medium overnight. Cells were then treated with assay buffer containing rolipram (10 mM) and antagonists for 20 min followed by the addition of agonists and incubate for 10 min. After 10 min incubation with agonist, Forskolin (10 μM) was added to the mixture and the incubation was continued for another 10 min. The reaction was terminated upon removal of the supernatant and addition of 100 µl Tween-20 (0.3%). Intracellular cAMP levels were measured with an ALPHAScreen cAMP assay kit as instructed by the manufacturer (PerkinElmer). Binding and functional parameters were calculated using Prism 7.0 software (GraphPAD, San Diego, CA, USA). Data were expressed as mean ± sem.

HEK293 cells stably expressing the human A_2AAR were seeded in 96-well plates and incubated in 100 µL medium at 37 °C overnight. The medium was removed the following day and cells were then treated with assay buffer containing rolipram (10 μM) and antagonists for 20 min followed by the addition of agonists, and then incubated for 20 min. The reaction was terminated upon removal of the supernatant and addition of 100 µL Tween-20 (0.3%). Intracellular 3′,5′-cyclic adenosine monophosphate (cAMP) levels were measured with an ALPHAScreen cAMP assay kit as instructed by the manufacturer (PerkinElmer, Boston, MA, USA).

HEK293-mGPRC6A cells were seeded at a density of 10⁵ cells per well into PDL-coated 96-well plates and incubated overnight in DMEM, supplemented with 10% FBS (v/v) at 37°C in 5% CO₂. Cells were then incubated in 90 μl of stimulation buffer (HBSS containing 5 mM HEPES, 0.1% BSA (w/v), 0.5 mM IBMX; pH 7.4) for 30 min at 37°C. Test compounds (10 μl at 10 x final) were added and cells were incubated for an additional 30 min at 37°C. The medium was removed and cells were incubated in 50 μl of ice-cold 100% ethanol. Once evaporated, 100 μl of lysis buffer (5 mM HEPES, 0.1% BSA (w/v), 0.3% Tween-20; pH 7.4) was added to the cells and samples were stored at -20°C until further analysis. Samples were analysed using the Alphascreen^® cAMP assay kit (Perkin Elmer) following manufacturer instructions. Briefly, 10 μl of lysates were added to a 384-well Optiplate. After addition of 5 μl of acceptor bead mix, plate was incubated at RT for 30 min followed by addition of 15 μl of donor bead mix. After overnight (16 h) incubation at RT, plates were read using the Envision plate reader.