Radioligand-binding studies

Opioid receptor expression was quantified by saturation binding assay using [³H]Diprenorphine (PerkinElmer, Boston, MA, USA) as we previously described (Chakrabarti et al. 2016). Briefly, cell membranes (50 μg/assay) were incubated with [³H]Diprenorphine (0.1–5 nM) in 50 mM Tris buffer at 25°C for 90min. Nonspecific binding was defined as that persisting in the presence of 10 μM DAMGO. Assays were terminated by the addition of ice-cold buffer and filtering over GF/B filters using a Brandel cell harvester (Brandel, Gaithersburg, MD). Liquid scintillation spectrophotometry (Beckman, Brea, CA, USA) was used to quantify radioactivity trapped on the GF/B filter.