Optimize, Adapt and Overcome - 34

Optimize, Adapt, and Overcome: Tips from the Cell Line Development Front * Screening and Selection of GPCR-Expressing Cell Lines

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.

C
Figure 3. The FLIPR® Tetra System performs high-throughput,
functional cell-based assays and is the system of choice in
drug discovery for evaluating changes in intracellular calcium
detected through the use of fluorescent calcium-sensitive
reporter dyes (FLIPR Calcium Assay 6 Kit).
34

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Optimize, Adapt and Overcome

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