CHO-K1 hCB1R and hCB2R cells were disrupted by cavitation in a pressure cell, and membranes were sedimented by ultracentrifugation, as described by Bolognini et al.34 (link),58 (link). The pellet was resuspended in TME buffer (50 mM Tris–HCl, 5 mM MgCl2, 1 mM EDTA, pH 7.4) and membrane proteins were quantified with a Bradford dye-binding method (Bio-Rad Laboratories, Mississauga, ON).
Assays were carried out with [3H]CP55,940 and Tris binding buffer (50 mM Tris–HCl, 50 mM Tris–base, 0.1% BSA, pH 7.4), total assay volume 2 mL, using the filtration procedure described previously by Baillie et al.18 (link). The binding was initiated by the addition of transfected human CHO-K1 hCB1R and hCB2R cell membranes (50 µg protein per well). All assays were performed at 37 °C for 60 min before termination by the addition of ice-cold Tris binding buffer, followed by vacuum filtration using a 24-well sampling manifold (Brandel Cell Harvester; Brandel Inc, Gaithersburg, MD, USA) and Brandel GF/B filters that had been soaked in wash buffer at 4 °C for at least 24 h. Each reaction well was washed 6 times with 1.2 mL aliquots of Tris-binding buffer. The filters were air-dried overnight and then placed in 5 mL of scintillation fluid (Ultima Gold XR, PerkinElmer). Radioactivity was quantified by liquid scintillation spectrometry. Specific binding was defined as the difference between the binding that occurred in the presence and absence of 1 µM unlabelled CP55,940. The concentration of [3H]CP55940 used in our displacement assays was 0.7 nM.
Assays were carried out with [3H]CP55,940 and Tris binding buffer (50 mM Tris–HCl, 50 mM Tris–base, 0.1% BSA, pH 7.4), total assay volume 2 mL, using the filtration procedure described previously by Baillie et al.18 (link). The binding was initiated by the addition of transfected human CHO-K1 hCB1R and hCB2R cell membranes (50 µg protein per well). All assays were performed at 37 °C for 60 min before termination by the addition of ice-cold Tris binding buffer, followed by vacuum filtration using a 24-well sampling manifold (Brandel Cell Harvester; Brandel Inc, Gaithersburg, MD, USA) and Brandel GF/B filters that had been soaked in wash buffer at 4 °C for at least 24 h. Each reaction well was washed 6 times with 1.2 mL aliquots of Tris-binding buffer. The filters were air-dried overnight and then placed in 5 mL of scintillation fluid (Ultima Gold XR, PerkinElmer). Radioactivity was quantified by liquid scintillation spectrometry. Specific binding was defined as the difference between the binding that occurred in the presence and absence of 1 µM unlabelled CP55,940. The concentration of [3H]CP55940 used in our displacement assays was 0.7 nM.
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