CB1 and CB2 functional assays Sample Clauses

CB1 and CB2 functional assays. 293T-CB1 and 293T-CB2 cells (stably transfected with CB1 and CB2 cDNAs) are incubated in 24-well plates (1x105 cell/ml) and transiently transfected with 0,1 mg/ml of the plasmid CRE-luc that contains six consensus cAMP responsive elements (CRE) linked to firefly luciferase. Transient transfections are performed with Rotifect (Xxxx Xxxx GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions and harvested 24 h after transfection. For agonistic activity of CB1 the cells are treated with the compounds for 6 h. For antagonistic activity of CB1 the transfected cells are incubated with increasing concentrations of the test compounds for 15 min and then with WIN-55, 212-2 (positive control for CB1), during 6 h and then luciferase activity measured in the cell lysates. Forskolin is an adenylate cyclase activator that is used at 10 mM as a positive control of cAMP signaling pathway activated by a CB1R-independent mechanism. For CB2 agonistic activity we treated 293T-CB2-CRE-luc cells either with increasing concentrations of the test substances or with WIN-55, 212-2 (positive control for CB2), for 15 min and then with Forskolin (10 mM). After six hours of stimulation the cells will be lysed in 25 mM Tris-phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol and luciferase activity measured using TriStar2 Berthold/LB942 multimode reader (Berthold Technologies) following the instructions of the luciferase assay kit (Promega, Madison, WI). The background obtained with the lysis buffer will be subtracted in each experimental value, and the specific transactivation expressed as fold induction over basal levels (CRE-Luc).
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