Colonies picked by the ClonePix 2 System were deposited into 96-well plates containing liquid expansion media, cultured for one week, and transferred to 384 well plates. Picked colonies were functionally corroborated with the FLIPR Tetra System using the FLIPR Calcium Assay 6 Kit. The kit measures transient intracellular calcium mobilization as a result of activation of the M1 G-protein coupled IP3 sensitive pathway. Carbachol was used as the muscarinic agonist.
In the high fluorescent clones (Figure 3A) picked by the ClonePix 2 System, carbachol produced a fourfold increase in fluorescent read from background. In the mixed medium and low (Figure 3B) fluorescent picked clones, carbachol produced a four- and twofold increase in fluorescent read from background, respectively. Finally in CHO-K1 negative control group (Figure 3C), carbachol failed to elicit any significant change in fluorescent read from background. Changes in baseline fluorescence intensity were normalized to background fluorescent reads of each 384-well cell plate before the addition of 40 nM carbachol.
These results support a positive correlation between membrane-bound G-protein coupled muscarinic receptor expression level and functional activity. The lack of calcium fluorescent signal in the CHO-K1 negative control group further confirms that ClonePix 2 System can accurately distinguish between clones that are positive or negative for cell surface expression. Relative differences in the expression of membrane-bound M1 GPCR resulted in commensurable fluorescence intensities as recorded for high, low, and no (negative control) M1 expression respectively.