| Assay Method Information | |
| | PRESTO-Tango Assay |
| Description: | HTLA cells (HEK-T cells stably expressing a β-arrestin2-TEV fusion protein and a tTa-dependent luciferase reporter) were maintained in DMEM (Corning) containing 10% FBS, 2 μg/ml puromycin and 100 μg/ml hygromycin B in a humidified atmosphere at 37 °C with 5% CO2. In brief, cells were plated to 50% confluency and transfected with a codon-optimized MRGPRX-Tango construct using the calcium phosphate method53. The next day, transfected cells were transferred to glass-bottomed, poly-L-lysine-coated white 384-well plates at a density of 20,000 cells/well. The following day, cells were treated with 10 μM concentrations of small molecules (in quadruplicate) diluted in drug buffer (1× HBSS with 20 mM HEPES and 0.3% bovine serum albumin, pH 7.4) and incubated for 18-24 h. After drug incubation, medium was removed and 20 μl of Bright-Glo (Promega; diluted 20-fold) was added to each well and incubated 15 min at room temperature. |
| Affinity data for this assay | |
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