| Assay Method Information | |
| | In Vitro Cannabinoid Receptor Activity Assay |
| Description: | Chinese Hamster Ovary (CHO) cells stably expressing either human CB1 or CB2 cDNA and the promiscuous G-protein Gαq16 were transferred to each well of a black Costar 96-well optical bottom plate (Corning Corporation). Each plate was incubated at 37 C. for 24 hr to confluence. The culture media was removed from the plates and cells were subsequently loaded with a fluorescent calcium probe (Calcium 5 dye, Molecular Devices) at a final loading concentration of 2 μM in a HBSS-based buffer containing 20 mM HEPES, 1% BSA and 10 μM Probenecid (Sigma) in a total volume of 225 μl. Cells were incubated at 37 C. for 1 hr and then stimulated with various concentrations of a test agent using a FLIPR Tetra plate-reader, which automatically added the agonist at 10 concentration to each well after reading baseline values for 17 sec. Agonist-mediated change in fluorescence (488 nm excitation, 525 nm emission) was monitored in each well at 1 sec intervals for 60 sec and reported for each well. Data were analyzed using Prism software (GraphPad). |
| Affinity data for this assay | |
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