Page 90 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

By enabling long term, continuous live-cell CMP — Cytotoxic mechanism CHX — Cytototatic mechanism
analysis under uninterrupted incubation, the
IncuCyte® S3 multi-spheroid assay makes it
possible to derive meaningful EC50 values
for drugs by deriving concentration response
curves over several days without disturbing
cell biology. In the figure below (Figure 7)
this is demonstrated for CMP and CHX, and
this approach is used to further validate
drug mechanism of action observed through
earlier analysis.

Figure 7. Determination of EC50s of CMP and CHX using the IncuCyte S3 Live-cell Analysis System to confirm mechanisms of action in response to cytotoxic
(CMP) and cytostatic (CHX) drugs. IncuCyte A549 NucLight Red Cells (2k/well, nuclear-restricted FB indicating viability) were seeded in the present of IncuCyte
V Annexin Green reagent (1%, apoptosis marker) and spheroids allowed to form for 3 days. Spheroids were then treated with a concentration range of either
CMP, CHX, or vehicle control, and the cells were imaged every 6 h for 7 d. Both CMP (cytotoxic) and CHX (cytostatic) drugs caused a concentration-dependent
inhibition of spheroid growth (total BF time courses, not shown). The CMP EC50, using AUC from 0-7 d after treatment, showed a concentration-dependent
loss of viability and increase in apoptosis, while CHX EC50s showed little change in viability and no increase in apoptosis.

Reproducible, quantitative
data at throughput suitable for
pharmacological testing

A pharmacological study performed in
MCF-7 breast cancer cells illustrates the
potential of the IncuCyte’s multi-spheroid
assays for drug toxicity testing (Figure 8,
below). The ability to view spheroids in
96-well plates over time provides a rapid
method for comparing the potencies and
toxicities of different compounds within
the same assay.

Figure 8. Effect of CMP, cisplatin (CIS) and
oxaliplatinin (OXA) on growth of MCF-7 cells in
a 3D spheroid assay. MCF-7 cells were plated at
a density of 1,000 cells per well and spheroids
allowed to form (72 h). Cells were then treated
with serial dilutions of compounds and the
kinetics of spheroid growth were obtained.
Plate view shows the individual well Total
Brightfield area (μm ) over time. Concentration
2
response curves represent the area under curve
of the Total Brightfield area time course (μm )
2
from 0 - 168 h post-treatment. Data were
collected over 168 h period at 6 h intervals.
Each data point represents mean ± SEM, n=3
wells.
88