Page 96 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Viability and toxicity measurements using non-perturbing
reagents enable investigation of drug mechanisms of action

Masking of the DF Brightfield channel allows measurement of Applying the ‘fluorescence within the Brightfield boundary’
other parameters of interest when non-perturbing reagents are feature in the IncuCyte® S3 spheroid software module negates
employed. In Figure 5 (below) the effects of CMP on cell viability the need to apply a fluorescence mask, and removes the impact of
were studied by stably expressing a red fluorescent protein (using threshold settings.
IncuCyte NucLight Red Lentiviral Reagent) in the spheroids.

Vehicle 1 µM CMP

Figure 5. Analysis of spheroids expressing fluorescent proteins enables
spheroid viability determination. Representative images taken at 240
h show a strong red fluorescent signal in a vehicle control spheroid, in
contrast to a marked loss in red fluorescence in the CMP-treated spheroid.
The yellow boundary in the images represents the Brightfield mask outline.
Monitoring the integrated intensity from within the Brightfield boundary
highlights a gradual increase in fluorescence under vehicle control
conditions (red symbols) corresponding to the growth of the spheroid. Upon
treatment with CMP, a concentration-dependent reduction in integrated
fluorescence is observed, with abolishment of fluorescence with the highest
concentration tested after 240 h.

A similar approach was used with IncuCyte® Cell Health Reagents analysis revealed a concentration-dependent effect with increases
to determine the mechanism of cell death after treatment with in the mean intensity within the Brightfield boundary, suggesting
CMP (Figure 6). Little or no reduction in spheroid size was observed induction of apoptosis.
with the Brightfield phase contrast images, but fluorescence

Vehicle 1 µM CMP

Figure 6. Effect of CMP on A549 cells reported by IncuCyte® Annexin V
Green reagents in a 3D spheroid assay. A549 cells were seeded at a density
of 2,500 cells per well in ULA round bottom plates and spheroids were
formed for 96h. Spheroids were treated with CMP (0.4 nM - 1 μM) or
vehicle (0.1% DMSO) and apoptosis was reported using IncuCyte Annexin
V Green.

94