CB2 Receptor Agonist Binding Assay

Ten micromolar
concentrations of each compound were incubated in triplicate with
membrane preparations from CHO-K1 cells expressing the human CB2 receptor
(0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an
assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA,
3 mM MgCl₂, 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA)
in the presence of 0.08 nM [³⁵S] guanosine 5′-[γ-thio]triphosphate
([³⁵S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).
Nonspecific binding was determined with 100 μM of unlabeled
GTPγS. CP-55,940 (100 nM) was used as stimulating ligand. The
final DMSO concentration in the assay was 5%. The reaction mixture
was incubated for 60 min at 30 °C. Next, the samples were deposited
under vacuum with the FilterMate Harvester (PerkinElmer, USA) onto
Unifilter GF/B Plates (PerkinElmer, USA) presoaked with wash buffer
(50 mM Tris–HCl, pH = 7.4). The samples were then rapidly washed
with 2 mL of wash buffer. Filter plates were dried for 30 min at 50
°C and 40 μL of MicroScint PS (PerkinElmer, USA) scintillation
fluid was added to each well. Radioactivity was counted in a Trilux
MicroBeta² counter (PerkinElmer, USA). Data were analyzed
with GraphPad Prism 5.0 software. Results were expressed as percent
of basal [³⁵S]GTPγS binding in the presence of CP-55,940
from three separate experiments. Basal binding was set to 100%.