β-Arrestin Recruitment Assay

The previously described protocol was followed.⁴⁶ In brief, HTLA cells, an HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene, were maintained in DMEM with 10% FBS, 2 μg/mL puromycin, and 100 μg/mL hygromycin B in a humidified atmosphere at 37 °C in 5% CO₂. For the transfection, cells were plated to 50–80% confluency and transfected with OPRM1 (for MOR) or OPRK1 (for KOR) using FuGENE HD (Promega Corp., Madison, WI, USA). The next day, transfected cells were trypsinized and transferred into poly-l-lysine coated and rinsed 96-well, white, clear-bottom cell culture plates in 50 μL of DMEM containing 1% dialyzed FBS and 1X penicillin/streptomycin at a density of 20 000 cells/well. After 7 h, 6X drug solutions were prepared in assay buffer (1X HBSS with 20 mM HEPES, pH 7.4), and 10 μL was added to each well. The following day, 60 μL of Bright Glo (Promega Corp.), diluted 4-fold, was added to each well and incubated 5 min at room temperature in the dark before luminescence was measured. Results in the form of luminescence units were analyzed using GraphPad Prism.