Optimize, Adapt and Overcome - 31

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

calcium changes. These typically require a much
higher concentration of functional GPCRs on the
cell surface than endogenous expression levels.
While a need exists for an expression system capable of supplying overexpressed GPCRs, it is important to also establish in vitro assays that mimic
the activity of endogenously expressed targets.
This challenge incites the need to utilize mammalian expression systems capable of providing a
range of requisite GPCR protein expression levels.

Identification and Selection of
GPCR Expressing Cell Lines
A solution utilizing an automated high-throughput platform that identifies and isolates heterogeneous expressing pools of cells provides significant value and saves considerable resource.
The ClonePix™ 2 System from Molecular Devices
represents a proven, one-step method of screening large cell populations rapidly. Utilizing white

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light and fluorescent images in situ, the ClonePix
2 System has the sensitivity to detect both overexpressed and endogenous levels of cell surface
protein expression.
The principle of ClonePix technology is based
upon plating single cells in a semi-solid matrix and growing into distinct clonal colonies.
A fluorescent antibody against the target cell
surface protein is also added; the antibody is
small enough to diffuse through the matrix and
bind to the cell surface. The physical location
and morphology of the colonies are identified in
white light; the corresponding fluorescent signal
is proportional to the level of expression on the
cell surface. The morphological and fluorescence
criteria can be refined before acceptable colonies
are picked into liquid expansion media. These
colonies can be scaled up to generate cell lines
or be utilized for further characterization studies.

Detecting Endogenous
Muscarinic 1 Receptor
A transfected CHO-M1 cell line expressing muscarinic 1 cholinergic receptor (M1) was chosen to
demonstrate the feasibility of using the ClonePix
2 System to detect cell surface proteins. CHO-M1
and parental untransfected CHO-K1 cells (negative
control) were plated in Molecular Devices CloneMedia CHO semisolid media at low density and
incubated until discrete colonies were formed.
Both a direct-labeled and a dual-label approach
were tested. For the direct-labeled approach,
a Phycoerythrin (PE)-labeled anti-M1 antibody
was used. For the dual-label approach, an
unconjugated rabbit anti-M1 antibody and a
PE-labeled anti-rabbit polyclonal antibody
were used. A dual label approach was done to
determine feasibility in cases where a labeled
primary antibody is unavailable.

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Optimize, Adapt and Overcome

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