Page 88 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Fluorescent readouts of stably expressing nuclear restricted fluorescent protein
act as a surrogate for cell viability

When a range of antibodies were tested against different cell surface markers in various
lymphocyte cell lines (Figure 4) it was found that the internalization patterns corresponded
with the known surface marker expression of these cells. Labeling antibodies with IncuCyte®
FabFluor-pH Red showed that anti-CD20 was internalized in a B-cell line, but not a T-cell line,
whereas anti-CD3 was internalized in T-cells and not B-cells. Antibodies to CD71 and CD45,
which are general lymphocyte markers, were internalized in both cell types. Crucially, the
non-specific mouse IgG was not internalized in any of the cell lines. These data demonstrate
the broad application and specificity across different cell types and molecules.

Figure 5. Analysis of spheroids expressing IncuCyte® NucLight Red fluorescent proteins enables determination of spheroid viability. Representative images
taken at 144 h show a strong red fluorescent signal in a vehicle control spheroid, in contrast to a marked loss of red fluorescence in the CMP treated spheroid.
The yellow boundary in the images represents the DF Brightfield mask outline. Monitoring the integrated intensity from within the DF Brightfield boundary
highlights increasing fluorescence under vehicle control conditions corresponding to the growth of the spheroid. Upon treatment with CMP (0.4 nM - 1
μM), a concentration-dependent reduction in integrated fluorescence is observed, with complete loss of fluorescence at the highest concentration tested
after 144 h.

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