| Solution Information | help | |
| Enzyme: | Muscarinic acetylcholine receptor M1 | |
| inhibitor: | BDBM43822 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | The purpose of this screen was to test compounds of interest against the muscarinic panel (M1, M2, M3, M4 and M5) for their ability to modulate calcium flux. Experimental Protocol: Stable CHO-K1 cell lines expressing rat M1, human M2-Gqi5, human M3, rat M4-Gqi5, or human M5 were plated into 96-well Costar optical bottom assay plates at a concentration of 50K cells/well in 100 microliters of Ham's F-12 media. Cells were incubated overnight at 37 degrees C (5% CO2). The following day, medium was removed from cells and they were incubated in 50 microliters of Fluo-4 AM diluted in assay buffer (HBSS containing 20 mM HEPES and 2.5 mM probenecid, pH 7.4) for 1 hour at 37 degrees C. Dye was removed and replaced with 45 microliters of assay buffer. Test compounds were serially diluted in DMSO-matched assay buffer at 2X concentration and 45 microliters was applied to a cell-containing assay plate in an automated format using the Flexstation (Molecular Devices). Calcium flux was meas | |
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