NVP-BKM120 1202777-78-3 age buffer, homogenized again, aliquoted and frozen at 801C

. Protein concentrations were determined using Bio Rad Protein Assay reagents as per the manufacturer,s instructions. Binding NVP-BKM120 1202777-78-3 assays were conducted using 30 mg, 50 mg or 12mg membrane protein per tube and 1 3 nM CP55,940 as the radioligand, NVP-BKM120 1202777-78-3 compounds were diluted to 10 concentrations in 4% DMSO/H2O, and all reagents were combined in the assay buffer. The assay was incubated at 301C for 60 min and filtered on Whatman GFB filter mats treated with 0.15% polyethyleneimine using a Brandel 96 channel harvester. Radio cAMP inhibition assays Cells cultured in T 175 flasks were harvested by washing twice with PBS, followed by addition of 5ml cell dissociation solution.
After 3 5 min buy NVP-BKM120 incubation at room temperature, the dissociated cells were removed, mixed with 10 ml Krebs assay buffer and pelleted.
Cell pellets were resuspended in Krebs and counted. Cannabinoid ligands were serially diluted in Krebs containing buy NVP-BKM120 1 mM forskolin. Per well of a 96 well plate, the ligand/forskolin mixture was combined with 1.5 104 cells and incubated at 371C for 30 min. cAMP determinations were performed using the HitHunter cAMP XS Assay according to the manufacturer,s protocol. Chemiluminescence was counted using aWallac Victor V after a 3h incubation. For the Pertussis toxin study, cells were incubated in the presence of 100 ngml 1 Pertussis toxin for 4h before forskolin stimulation.
In vivo studies All animal procedures were approved by an institutional animal care and use committee and were conducted in accordance with the International Association for the Study of Pain guidelines on the use of animals in experimental research.
Acute analgesia was investigated using the tail flick and hot plate assays. For the tail flick assay, male Sprague Dawley rats were placed on the apparatus, and an infrared beam was focused 5 cm from the tip of the tail. The latency to tail flick was measured to the nearest 0.1 s with a cutoff of 20 s. For the hot plate assay, male Sprague Dawley rats were placed on a metal plate maintained at 521C. The latency to nocifensive response, defined as hindpaw lift, flutter, licking or escape behaviour, was measured to the nearest 0.
1 s with a cutoff of 30 s. Approximately, 1 h after determination of baseline latency, animals received a single intraperitoneal dose of vehicle or 1, 3 or 10mgkg 1 R,S AM1241, R AM1241 or S AM1241.
Dosing of the positive control was by subcutaneous injection. Tail flick and hot plate latencies were determined 30 and 90 min after drug administration. The ability of compounds to attenuate painful abdominal stretching was assessed in male CD 1 mice following i.p. injection of 2mg kg 1 paraphenylquinone . Delivery of R,S AM1241, R AM1241 or S AM1241 was as a suspension in vehicle 30 min before PPQ injection. Following PPQ administration, mice were placed individually in a Plexiglas observation cage, and stretching movements were recorded for two periods of 1 min each, at 5 and 10 min post injection. Percent blockade was calculated according to the following equation: % Blockade meanvehicleT emeandrugT emeanvehicleT 1 100% Latency of paw withdrawal from a thermal stimulus was assessed in male Sprague Dawley rats in response to focusing a radiant heat source on the pl