2.6. cAMP accumulation assay

cAMP accumulation assay was performed using the LANCE Ultra cAMP competitive immunoassay kit (PerkinElmer, Waltham, MA). Briefly, N2A-D2R cells suspended in HBSS buffer (5 mM Hepes, 0.5mM IBMX and 0.1% BSA) were seeded onto 384-well white, opaque OptiPlates (Perkin Elmer). Forskolin (10 μM) and quinpirole (0.1 nM-1 μM) were added to the wells and incubated at room temperature for 30 mins. Next, Eu-cAMP tracer and ULight-anti-cAMP were added to the mixture which was then incubated for 1 hr at room temperature. Plates were read for TR-FRET signal using the Victor3 plate reader (Perkin Elmer) with 340 nm wavelength excitation and 665 nm wavelength emission. The cAMP standard curve was fitted as a sigmoidal 4PL (X, log(agonist)) equation in Prism (GraphPad Software) with the corresponding equation: Y = bottom + (top − bottom)/(1 + 10^{^}[(LogEC50 − X) * Hill slope]). The true cAMP concentration in each sample was extrapolated from the cAMP standard curve with X representing log [cAMP] and Y representing TR-FRET signal emitted at 665 nm. Data were presented as percent of the maximal cAMP accumulation produced by stimulation with forskolin. The values of IC50 were determined.