| Solution Information | help | |
| Enzyme: | Muscarinic acetylcholine receptor M5 | |
| inhibitor: | BDBM50258656 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Info: CHO-K1 cells stably expressing human M5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37 degrees 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 diluted in assay buffer for a 60 uM 2X stock concentration (30 uM final concentration) in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine CRC serial dilutions were 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 were then stimulated for 50 sec with a one of eight concentrations of the | |
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