CHO-hKOR cells (15,000 cells per well) were plated in a clear-bottom black 384-well plate (BD Falcon) with DMEM/F12 medium containing 10% FBS with or without pertussis toxin (100 ng/ml) for overnight. The cells were then incubated with 1× HBSS (Hanks’ balanced salt solution)/20 mM Hepes at 37°C for 2 hours followed by calcium-sensitive dye loading along with 125 mM probenecid for 1 hour at 37°C. The KOR agonist–induced intracellular calcium mobilization was determined by using a FLIPRTETRA instrument to measure fluorescence intensity [excitation/emission (Ex/Em) = 490 nm/515 nm].

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