Calcium Mobilization Assay for Opioid Receptors

Stock solutions (10 mM) of the peptides were prepared in 5% DMSO in bidistilled water and kept at −20°C Serial dilutions were carried out in HBSS/HEPES buffer (20 mM, containing 0.02% bovine serum albumin fraction V). Calcium mobilization assay was performed using the same method as previously described (Camarda and Calo, 2013 (link)). CHO cells with stable co-expression of human mu or kappa receptors and the C-terminally modified Gα_qi5 and CHO cells with co-expression of the delta-opioid receptor and the Gα_qG66Di5 protein were used. Dulbecco’s MEM/HAMS F12 (1:1) medium supplemented with 10% fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), L-glutammine (2 mM), fungizone (1 μg/mL), geneticin (G418; 200 μg/mL) and hygromycin B (100 μg/mL) was used for cell culture. Cells were seeded at a density of 50,000 cells/well into 96-well black, clear-bottom plates and kept in the incubator at 37°C in 5% CO₂/humidified air. After 24 h the cell growth medium was aspired and loading medium, supplemented with probenecid (2.5 mM), calcium sensitive fluorescent dye Fluo-4 AM (3 µM), pluronic acid (0.01%) and HEPES (20 mM), was added. Then the plates were placed in the incubator again. After 30 min the loading solution was aspirated and 100 µL/well of assay buffer (HBSS supplemented with 20 mM HEPES, 2.5 mM probenecid, and 500 µM Brilliant Black) was added. Next, both plates (cell culture and compound plate) were placed in the FlexStation II reader (Molecular Device, Union City, CA 94587, United States), the on-line additions were carried out in a volume of 50 µL/well and the fluorescence changes were measured. Ligand efficacies, expressed as the intrinsic activity (α), were calculated as the E_max ratio of the tested compound and the standard agonist. At least three independent experiments for each assay were carried out in duplicate.
Curve fittings were performed using Graph Pad PRISM 5.0 (GraphPad Software Inc., San Diego, United States). Data have been statistically analyzed with one way ANOVA followed by the Dunnett’s test for multiple comparisons; p values < 0.05 were considered significant.