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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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6X Loading Buffer is intended for gel electrophoresis of DNA fragments or PCR products....
6X DNA Gel Loading Buffer is intended for gel electrophoresis of DNA fragments or PCR products....
Makes gels easy to load and track during electrophoresis. 10X concentrate of Bromophenol Blue, Xylene Cyanol, glycerol solution in TE...
Agarose Gel Loading Dye Mixture 6X...
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