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 FLIPR^TETRA. 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.