| Solution Information | help | |
| Enzyme: | Muscarinic acetylcholine receptor M4 | |
| inhibitor: | BDBM48075 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | The purpose of this assay was to test the lead compound for potency against rM4/Gqi5 in a calcium mobilization assay. Assay Info: CHO-K1 cells stably expressing rat M4 and Gqi5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37degrees C. Dye was removed and replaced with the appropriate volume of assay buffer, pH 7.4 (1X HBSS (Hanks' Balanced Salt Solution), supplemented with 20 mM HEPES and 2.5 mM probenecid). All compounds were serially diluted in assay buffer for a final 2X stock in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine EC20 was prepared at a 10X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 25 degrees C using a FLEXstation II (Molecular Devices, Sunnyvale, CA) according to the following protocol. Cells were preincubated with test compound (or vehicle) for 1.5 min prior to the addition of the agonist, acetylcholine. Cells | |
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