Optimize, Adapt and Overcome - 15

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

colonies have formed about 6 to 10 days postseeding. The use of fluorescence also enables
accurate determination of cell counts based on
thresholding, automating an otherwise laborious
process (Figure 2). Finally, one can use fluorescence probes that only stain viable cells, eliminating the risk of dead cells, debris, or well artifacts
from erroneously contributing to cell count. A
white light overlay provides added assurance that
what is detected in fluorescence in fact appears
as a cell in brightfield.

Automated cell counting per well
using fluorescence for monoclonality
identification
Detection of fluorescence signal from stained
cells enables the automated detection and count
of cells in an objective and reproducible manner.
Single-cell wells are marked green while multiple
cell-containing wells are marked red. Dividing
cells are marked yellow based on a pre-specified
x and y-axis symmetry ratio while void wells are
labeled white. Example images of each classification type are shown in Figures 2 B-E.
15

| GENengnews.com

Results
Using the fluorescence workflow to
determine the optimal Calcein-AM
concentration
A critical parameter in developing a reliable labeling protocol prior to limiting dilution is ensuring
that the cells are adequately labeled for detection
using the fluorescence channel on the CSI. An
ideal staining condition would have reproducible
low concentration sufficient to label all or most
cells without the cytotoxic effects as single cells
proliferate to form colonies.
Three concentrations of CAM viability dye were
compared under identical staining conditions.
The concentrations tested were 5 µM as a high
staining range, 1 µM as a medium level stain and
0.5 µM as a low-level stain. To confirm that the
staining method works, we set up an initial staining~1000 cells per well using the three concentrations of dye. We scanned those wells using
fluorescence and brightfield on the CSI under
identical exposure well is shown in Figures 3A, B,
and C. One can observe that the majority of cells

are detectible at the 5 µM (Figure 3A) and 1 µM
CAM conditions (Figure 3B), but not at the 0.5 µM
CAM condition (Figure 3C).
We then performed a limiting dilution at
0.5 cell per well using the stained cells then
scanned the plates in fluorescence and brightfield using the CSI under identical exposure
settings. The fluorescence- based cell count
automatically calculates the number of cells per
well, as shown in Figures 3D-F. From this, one
can observe that the 5 µM and 1 µM CAM conditions appear to show a similar distribution
of cells per well from limiting dilution (Figure
3D-E). Meanwhile, the 0.5 µM CAM condition
shows only a small number of wells containing
single and multiple cells (Figure 3F). This low
number of cells detected is consistent with the
notion that using 0.5 µM CAM is not sufficient
to stain the majority of the cells of interest to
a detectible level on the CSI. It is important to
note that colonies still formed in this calcein
plate (data shown in Figure 4), indicating that
cells were present, but not detectable with
fluorescence.

http://www.genengnews.com http://www.GENengnews.com

Optimize, Adapt and Overcome

Table of Contents for the Digital Edition of Optimize, Adapt and Overcome