Gain Critical Insights from Advanced Cell Models with Real-Time Analysis - 6

Real-Time Analysis of Advanced Cell Models | A CELLULAR JOURNEY BEGINS

croscope. It is difficult to get consistent reliable data. The IncuCyte Scratch Wound
Assay automates the process in a 96-well, high-throughput assay. An IncuCyte
WoundMaker is used to create 96 precise, uniform, cell-free zones in cell monolayers. Wound closure is then visualized and analyzed in real-time.

Manual angiogenesis assays also have hurdles. The IncuCyte angiogenesis kit uses
a co-culture system. GFP-labeled human umbilical vein epithelial cells (HUVEC)
are grown on top of human fibroblasts simulating an environment closer to
physiological angiogenesis where cells are in touch and surrounded by other cell
types. Green fluorescence monitors tube formation. Software parameters allow the
exact quantitation of many different angiogenic factors in the same experiment,
including tube formation, network and loop length.
Migrating from End-Point to Live-Cell Imaging Experiments
Some experiments, such as cell health or morphology, can be simple to transition
to live-cell imaging, while other experiments investigating functional aspects of
the cell may be more complex.
"An assumption may be considered true, as proven with single time points, but
the kinetic situation may reveal something different; for instance, a living cell
system may respond quickly or slowly to a given treatment. If compound A acts
fast, and compound B acts more slowly but causes more impact, can you judge the
difference if you analyze at a single timepoint? This does not imply that the original
data are wrong, but rather that they do not tell the complete story. More work may
be needed to better understand the biology and conditions before the hypothesis
can be proven," says Kim Wicklund, Ph.D., director of product management,
Sartorius.
"This is where the scale of the IncuCyte plays a major role and easily accommodates testing of multiple variables. As an example, induced pluripotent stem cells
offer great promise; however, precise control over differentiation and maintenance
of these cells is extremely important. Appropriate morphology may be present but
the necessary function may not. Careful characterization of these cells mandates
use of a system that enables evaluation of multiple media formulations or coatings
on both cell health and function, while perturbing the cells and their environment
as little as possible."
6

| January, 2019

Measurement of the reproducibility
the migration and invasion assay
in the same microplate. HT - 1080
cells were placed at a 2 x 104 cells
per well on a 100 μg/ml Matrigel
coated ImageLock plates. The cells
in odd numbered columns had
only media added after using the
96-well WoundMaker representing
cell migration. The cells in even
numbered columns were overlaid
with 8 mg/mL Matrigel representing
invasion. (A) Temporal progression of
wound closure in each well with time
using RWD as the metric to measure
migration or invasion. (B) Time course
of means of each condition. The
respective coefficients of variation
for each assay were averaged. (C)
Representative images of HT - 1080
cells migrating on Matrigel (left) and
invading through 8 mg/mL.

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