βarrestin2 Recruitment Assays

Qualitative βarrestin2 (βarr2) recruitment was measured using MOR- and D0R-βarr2EGFP-U20S cells.⁶ The assays were performed as described in ⁴³. The cells were plated onto bovine collagen type I-coated glass bottom microscopy dishes, and recovered overnight. The cells were serum starved for 1 hour in phenol red- and serum-free culture media. The cells were live-imaged with a Leica SP6 confocal microscope before and 5 minutes after addition of 10 μM of drug. βarr2 recruitment was visualized by the formation of bright green punctae. For the MOR-DOR co-expressing CHO cells, the cells were electroporated with 10 μg per cuvette of βarr2-EGFP DNA vector using a BioRad (Hercules, CA) Gene Pulser XCell, followed by plating, recovery, and imaging as above.

For the quantitative βarr2 recruitment assay using MOR- and DOR-U20S PathHunter cells from DiscoveRx, the manufacturer’s protocol was followed for all steps. The cells were treated with varying concentrations of agonist in 384 well black walled, clear bottomed plates, and the data collected using a BioTek (Winooski, VT) Synergy plate reader. The data were normalized to the stimulation caused by positive control agonist (100%; DAMGO for MOR and SNC80 for DOR) and vehicle (0%). Potency (EC₅₀) and efficacy (E_MAX) values were calculated after a 3 variable curve fit using Prism 7.0 (GraphPad).