MgCl2, and 0.05% fatty were terminated by rapid vacuum filtration through UniFilter 96 GF/C filter plates Tosedostat LPA receptor inhibitor Tosedostat LPA receptor inhibitor and four washes with cold assay buffer. Competition experiments were conducted using 0.5nM CP 55,940 in the presence of test compounds. Nonspecific binding was defined by 10 mM unlabeled CP 55,940. KD values from saturation binding assays and Ki values from competition binding assays were determined with one site binding or one site competition curve fitting using the Prism software. FLIPR assays were performed using HEK cells stably expressing the human CB2 receptor and chimeric Gaq/o5 protein with modification. Briefly, cells were seeded at 7.
5 104 cells per well in Biocoat Tosedostat 238750-77-1 96 well poly Llysine coated clear bottomed black wall plates 1 day prior to the assay.
The cells were incubated with No Wash Dye following vendor,s instruction. For agonist assays, variable concentrations of test compounds, CP 55,940 positive control Tosedostat 238750-77-1 or vehicle negative control were added to cells in the presence of assay buffer, and maximum fluorescence responses were measured with a FLIPR machine immediately following addition of compounds. Agonist responses were adjusted for the fluorescence background with vehicle controls and the activities were expressed as percentages of the CP 55,940 response.
For antagonist assays, vehicle or variable concentration of test compounds were added to the cells at the first addition and CP 55,940 was added to all cells at the second addition. The interval between the first and the second additions was 5 min.
For antagonist assays, the maximum fluorescence response was measured immediately after the addition of CP 55,940 at the second addition and the response was compared with the control where vehicle instead of test compounds was added at the first addition. EC50 and Kb values were analyzed with sigmoidal dose response curve fitting using Prism. The cyclase functional assays were performed using the HitHunter cAMP assay kit according to vendor,s protocols. Briefly, HEK cells expressing the human CB2 receptor were detached using cell dissociation buffer, dispersed and placed in suspension at 104 cells per well in 96 well plates prior to the assay.
For agonist and inverse agonist assays, cells were treated for 20 min with variable concentrations of test ligands and forskolin in Dulbecco,s phosphate buffered saline supplemented with BSA.
In experiments assessing the antagonist properties of AM1241, variable concentrations of AM1241 and forskolin were added to the cells together with a fixed concentration of either CP 55,940 or SR144528. The concentration of forskolin used to stimulate the cAMP level in cyclase assays was 37 mM unless indicated otherwise. Reactions were incubated for 20 min at 371C and terminated by the addition of lysis buffer and the luminescence was detected following the procedure according to vendor,s instructions. The cyclase activities were expressed as percent responses over the forskolin stimulated control levels, where cells received vehicle instead of test compounds. EC50 values were calculated using sigmoidal dose response curve fitting from Prism. HEK cells stably expressing the human CB2 receptor were seeded at 2 105 cells per well in six well plates,