| Solution Information | help | |
| Enzyme: | Muscarinic acetylcholine receptor M1 | |
| inhibitor: | BDBM65915 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | CHO-K1 cells stably transfected with rat M1 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37 degrees C. Dye was removed and replaced with 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 (ACh) submax concentration (~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 were then stimulated for 50 sec with a submaximal ACh concentration (~EC20). The signal amplitude was first normalized to basel | |
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