cAMP measurements in HEK293T cells
LANCE Ultra cAMP assays (Perkin Elmer; Waltham, MA) were performed in accordance with the manufacturer’s instructions. Briefly, the day before the assay, HEK293T cells were transfected as indicated above. On the day of experiment, cells were dissociated from dishes using Versene 1M. Then cells (2,000 cells/well in 5µl) were transferred to a white 384-well microplate (Perkin Elmer) and incubated with various concentrations of N/OFQ and SKF-38393 (D1 receptor agonist; final 40 nM; 5 µl/well) for 30 min at room temperature. A cAMP standard curve was generated in triplicate according to the manual. Finally, europium (Eu)-cAMP tracer (5 µl) and ULight™-anti-cAMP (5 µl) were added to each well and incubated for 1 h at room temperature. The plate was read on a TR-FRET microplate reader (Synergy NEO; Biotek, Winooski, VT).