Optimize, Adapt and Overcome - 12

Optimize, Adapt, and Overcome: Tips from the Cell Line Development Front * Confident Assurance of Clonality Using Calcein AM with Minimal Effect on Viability

identification of a single on day 0 is not without
challenge, as cellular debris and well artifacts
can be easily mistaken for cells. Consequently,
cell line developers typically evaluate cells at the
colony stage and trace back the origins of the
colony to confirm monoclonality.
An alternative method to assessing clonality involves fluorescently labeling the cell population
prior to seeding single cells. Here, we outline the
differences between a transmitted white light
(WL) workflow and one involving both WL and
fluorescence (FL). We primarily focus on the benefit of using fluorescence dyes to more easily
automate single cell detection while simultaneously establishing clonality more conclusively.
There are additional fluorescence techniques (GFP
expression systems, for example), which have further benefits, but are not discussed in detail here
because their workflows are very similar. However, when considering a fluorescence expression
workflow, it is important to evaluate the sensitivity of the imaging instrument as dyes tend to be
much brighter than expression-based approaches. In this application note, we use the viability
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dye calcein AM (CAM), a molecule that fluoresces
green only after translocating the membrane of
living cells. We lay out a basic approach to label
cells for reliable detection of single cell wells
while maintaining high rates of viability.

Materials and methods
Using calcein AM dye to label cells
before limiting dilution
In order to develop a workable cell staining protocol, we used the EarlyTox™ Cell Viability Assay
Kit. In this kit, calcein AM (CAM), a small cell-permeable molecule, can be employed as marker for
cell viability. Once CAM translocates the plasma
membrane, the AM moiety is cleaved by esterases in the cytosol, resulting in a green fluorescent calcein molecule. Calcein presence in the
cytosol is transient and viable cells typically eject
this molecule gradually over a span of hours.
Labeling cells and preparation
for limiting dilution
For limiting dilution, CHO-K1 cells (ATCC) were
used. CHO-K1 cells were grown in adherent con-

ditions in 35-mm dishes to 50-75% confluence.
To prepare for labeling, cells were washed with
PBS then incubated with serum-free media containing the specified calcein AM fluorescent dye
concentration for 40 minutes at 37°C. Cells were
then washed 3 times with PBS and lifted using
trypsin and prepared for counting. To eliminate
clumping, cells were filtered through a 40-µm
strainer prior to counting (Countess, ThermoFisher
Scientific). Cells were resuspended to 1 x 106
cells/mL in serum medium (10% FBS) and diluted
sequentially to achieve a 0.5 cells/well concentration and plated in 384-well plates.
Scanning plates in WL and FL using
the CloneSelect Imager (CSI)
Plates were imaged on a CSI modified to include
fluorescence capability. Imaging occurred within
30 minutes of seeding to allow cells to settle and
adhere to the well bottom. Microplates were
imaged using the combined fluorescence and
brightfield mode. Fluorescence images were
captured with a 300 ms exposure. A positive
control well containing 1000 cells as used to optimize focal height prior to plate scan, and the cell

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Optimize, Adapt and Overcome

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