BRET Quantification of Ligand Binding

Drug-induced BRET is conducted as optimized previously (i.e., transfection and experimental conditions) (Urizar et al., 2011 (link); Yano et al., 2018 (link)). Briefly, cells were prepared in 96-well plates. 5 μM coelenterazine h was added to each well. Three minutes after addition of coelenterazine h (Nanolight), ligands [(+)-pentazocine (Sigma), PD 144418 (Tocris), and haloperidol (Tocris)] were added to each well in serial dilution. BRET was measured as a ratio between measurements at 535 nm for fluorescence (gain 2500, interval time 0.9) and at 485 nm for luminescence (gain 2500, interval time 0.9) using a PHERastar FSX reader (BMG). Results are calculated for the BRET change (BRET ratio for the corresponding drug minus BRET ratio in the absence of the drug). E_max values are expressed as the basal subtracted BRET change in the dose-response graphs.