2.6. Cannabinoid Receptor Binding Assay

Receptor binding assay was performed on viable AEC, incubated as described above with the CB1/CB2 radiolabelled agonist [³H]CP55,940 (Perkin-Elmer Life Sciences, Boston, MA, USA) at different concentrations (0.5, 1, and 2.5 nM) in the presence of 50 mM Tris-HCl, 1 mM CaCl₂, 5 mM MgCl₂, and 0.2% BSA (Bovine Serum Albumin), pH 7.4 at 37 °C for 1 h. After this, the incubation buffer was removed and the cells were washed twice with 1 mL of ice-cold washing solution (50 mM Tris-HCl with 1% BSA). Subsequently, 500 μL of 0.1 M NaOH was added to each sample, and the cells were collected and transferred into a scintillation tube to test radioactivity. In all binding experiments, nonspecific binding was determined in the presence of 1 μM “cold” agonist CP55,940.