384 well white clear bottom cell culture plates

HTLA cells, (an HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) were a gift from the lab of Richard Axel, and were maintained in DMEM supplemented with 10% FBS, 2 μg/ml puromycin and 100 μg/ml hygromycin B in a humidified atmosphere at 37°C in 5% CO₂. For transfection, cells were plated at 9 to 10 × 10⁶ cells per 150 mm cell culture dish (day 1). The following day (day 2), cells were transfected using the calcium phosphate method. On day 3, transfected cells were transferred at 15,000 to 20,000 cells per well in 40 μl of medium into poly-L-lysine coated and rinsed 384-well white clear-bottom cell culture plates (Greiner Bio-one). On day 4, 3.5x drug stimulation solutions were prepared in filter-sterilized assay buffer, which consisted of 20 mM HEPES and 1x HBSS at pH 7.4, and 20 μl added to each well. On day 5, medium and drug solutions were removed from the wells (by aspiration or shaking), and 20 μl per well of Bright-Glo solution (Promega) diluted 20-fold in assay buffer were added to each well. After incubation for 15 to 20 minutes at room temperature, luminescence was counted in a Trilux luminescence counter. Results in the form of RLU (relative luminescence units) were exported into Excel spreadsheets, and Graphpad Prism was used for analysis of data. To measure constitutive activity, no ligand was added on day 4.