Hank s balanced solution

β-arrestin2 recruitment to the receptor was measured using the previously described Tango assay (Barnea et al., 2008 (link)). HEK-293T cells stably expressing the β₂V₂R tethered to the tetracycline transactivator transcription factor by a tobacco etch virus protease cleavage site, the human β-arrestin2 protein fused to the tobacco etch virus protease, and the tetracycline transactivator–driven luciferase reporter were used for this assay. Cells were plated on a 96-well, white clear-bottom plate at a density of ∼50,000 cells/well and were given at least a 24-hour incubation at 37°C, 5% CO₂, and ∼100% relative humidity to recover surface receptor expression. Cells were treated with either a varying dose of Cmpd-6 or a vehicle control (DMSO) diluted in Hanks’ balanced solution (Sigma-Aldrich), supplemented with 20 mM HEPES, pH 7.4, and 0.05% BSA, and then incubated at 37°C, 5% CO₂, and ∼100% relative humidity for ∼20 minutes. After the incubation, a serial dilution of the β-agonist was added, following which the cells were incubated for 6 hours at 37°C and ∼100% relative humidity. At the end of the incubation, the plate was cooled to room temperature. After adding the Bright-Glo reagent (Promega), chemiluminescence signals were read using a NOVOstar microplate reader (BMG Labtech) at 5–10 minutes.

Folin–Ciocalteu’s reagent, Hank’s balanced solution, Eagle’s minimum essential medium, and fetal calf serum were purchased from Sigma-Aldrich (Bengaluru, India).