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Gq-coupled receptor assays using FLEXstation: M1 muscarinic receptor

090). Cells were incubated either for one hour with a buffer containing 4.2 M Fluo-3 or for one-half hour with 1X Loading Buffer from the FLEXstation Calcium Assay Kit (Molecular Devices, cat#R8041) before using FLEXstation to dispense carbachol and monitor the mobilization of calcium into the cell cytosol. All experiments were performed at a final concentration of 2.5 mM probenecid in order to inhibit endogenous efflux pumps present in M1 CHO cells.


DETERMINATION OF EC50 VALUES
Cells were exposed to a range of carbachol (0 to 1 M) using half log dilutions of the agonist. Results are representative of at least four independent experiments performed over a period of at least two different days. The raw data was analyzed as Max-Min and Absolute RFU values (Figures 1, 2). The average value of replicate samples was plotted on a graph using a curve fit of four parameter, for which the C value is the concentration of drug that causes 50% of maximal response (EC50 value, see Figure 3). The EC50 values for carbachol using Fluo-3 ranged from 1939 nM, whereas it ranged from 1230 nM using the FLEXstation Calcium Assay Kit (see Figures 3 and 4).



DETERMINATION OF Z FACTOR
Cells in half of the microplate were treated with 0.5 M carbachol, while cells in the other half of the plate were treated with 1X Reagent Buffer (1X HBSS/20 mM HEPES pH 7.4). The FLEXstation Calcium Assay Kit was used to monitor carbachol-mediated calcium mobilization (Figure 5). Z factor was calculated using the formula2:

The terms sc- and sc+ denote the standard deviation of the negative control and positive control, respectively, whereas |c+ c-| denotes the absolute value of the difference in the mean of the positive control
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