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).