Page 38 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Kinetic Cytotoxicity Assays

Real-time, live-cell assay to quantify and visualize cytotoxic events

Introduction
IncuCyte Cytotoxicity Assay at a Glance
®
The cellular response to cytotoxic exposure is controlled by
complex biochemical pathways, such as necrosis or apoptosis, The IncuCyte Cytotoxicity assay uses highly sensitive cyanine
which results in cell death. In apoptosis, morphological changes nucelic acid dyes ideally suited for mix-and-read kinetic
include pseudopodia retraction, reduction of cellular volume measurements of cell membrane integrity overtime. IncuCyte
®
(pyknosis), nuclear fragmentation (karyorrhexis) and eventually CytoTox Red and Green Reagents are cell impermeant cyanine
loss of plasma membrane integrity. Morphological changes that dimer nucleic acid stains that bind to dsDNA,5 and when added to
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characterize necrosis include cytoplasmic swelling and early the culture medium, these reagents fluorescently stain the nuclear
rupture of plasma membrane. Compounds that have cytotoxic DNA of cells that have lost plasma membrane integrity.
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effects often compromise cell membrane integrity regardless of
the pathway. This cytotoxicity assay can be combined with IncuCyte® NucLight
cell labeling reagents that incorporate a red or green nuclear
Assays designed to measure cytotoxicity in vitro are used label allowing for simultaneous measurement of proliferation and
to predict tissue-specific toxicity or to identify and classify cytotoxicity in a single well. Non-perturbing NucLight reagents
leads for anti-cancer therapies. Multiplexed, high-throughput provide a means to kinetically quantify cell proliferation over time.
screening (HTS) in vitro cytotoxicity assays measuring a variety of The reagents allow for the expression of a nuclear-restricted GFP
different readouts are being employed to assess the cytotoxicity (green fluorescent protein) or mKate2 (red fluorescent protein) in
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of compounds in early drug development. Commonly used mammalian cells without altering cell function and with minimal
cytotoxicity assays evaluate a range of end-point parameters, such toxicity. NucLight lentivirus reagents allow for the creation of stable
as the release of lactate dehydrogenase (LDH) and glutathione cell populations or clones.
(GSH) following membrane rupture, generation of reactive oxygen
species (ROS), cell proliferation, and disruption of mitochondrial Phase-contrast images can be used to qualitatively monitor
trans-membrane potential. Critical factors contributing to the associated morphological changes in the same cells over the same
predictive nature of these assays include compound concentration, time course.
and more importantly, the time allowed for the compound to
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elicit an effect. Although these multiplexed assays are able to
simultaneously measure multiple indicators of in vitro cytotoxicity,
they typically assess a single time point and are unable to assess
the biological activity over time.

In this chapter, we will examine kinetic approaches for measuring
cytotoxicity using reagents that fluorescently stain the nuclear
DNA of cells that have lost plasma membrane integrity. Unlike
traditional endpoint approaches, kinetic live-cell imaging allows
for the analysis of time-dependent variation in treatment response
as well as the ability to differentiate between cytostatic and
cytotoxic effects.

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