Figure 1. Carbachol-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/M3 cells. The cells were loaded with Calcium-4 prior to being stimulated with an M3 receptor agonist, Carbachol. The intracellular calcium change was measured by FLIPRTETRA. The relative fluorescent units (RFU) were normalized and plotted against the log of the cumulative doses (10-fold dilution) of Carbachol (Mean ± SD, n = 2). The EC50 of Carbachol on this cell was 12.4 nM.
Notes:
1. EC50 value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom)/ (1+10^ ((LogEC50-X)*HillSlope))
X is the logarithm of concentration. Y is the response
Y is RFU and starts at Bottom and goes to Top with a sigmoid shape.
Figure 2 10 μg of membranes prepared from CHO-K1 cells stably expressing M3 receptors were incubated with indicated concentrations of [3H]N-Methylscopolamine ([3H]NMS) in the absence (total binding) or presence of 1000-fold access unlabeled Atropine (nonspecific binding, NSB). Binding was terminated by rapid filtration. Specific binding was defined by subtracting NSB from total binding. Data were fit to one-site binding equation using a non-linear regression method.
Figure 3 10 μg of membranes prepared from CHO-K1 cells stably expressing M3 receptors were incubated with indicated concentrations of Atropine in the presence of 0.2 nM [3H]N-Methylscopolamine ([3H]NMS). Binding was terminated by rapid filtration. Data were fit to one-site competition equation using a non-linear regression method.
CHO-K1/M3 Stable Cell Line
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