Page 31 - Live-cellanalysis handbook

Kinetic Caspase-3/7 and Annexin Apoptosis Assays

Live-cell Apoptosis Assays at a Glance

The IncuCyte® Caspsase-3/7 reagent is an inert, non-fluorescent • Multiplexing IncuCyte apoptosis reagents with a nuclear-
substrate. When added to tissue culture growth medium, the labeling reagent such as IncuCyte NucLight enables
substrate freely crosses the cell membrane where it is cleaved differentiation between inhibition of cell growth and induction
by activated caspase-3/7 resulting in the release of the DNA dye of cell death.
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and green fluorescent labeling of DNA. IncuCyte® Annexin V • High definition phase contrast images provide an additional
reagents are specially formulated, highly-selective cyanine-based qualitative validation of cell death based on morphological
fluorescent dyes ideally suited to a simple mix-and-read, real-time characteristics.
quantification of apoptosis in living cells.
• Images are automatically acquired and analyzed to reveal
• The apoptotic signal relies on either the activation of concentration and time-dependent effects on biology.
Caspase-3/7, a primary and irreversible “executioner” pathway
in most cell types, or using Annexin V conjugated to a
fluoroprobe for detection of PS externalization.
• Multiplexing the Caspase-3/7 and Annexin V reagents detect
and confirm apoptosis through two different pathways,
verifying apoptosis as a mechanism of cell death.

Shortcomings of Traditional Assays Live-Cell Imaging and Analysis Approaches

• Assays result in a single, user-defined time • The assay provides a full kinetic readout of apoptotic signaling over multiple
point measurement of caspase-3/7 activity. days, eliminating the need for determining a single, optimum, assay endpoint
a-priori which can vary considerably for different cell types and for different
• Techniques require multiple wash steps or compound treatment conditions.
cell lifting prior to data collection that may
result in the loss of dying cells or lead to a loss • Cells can be simultaneously labeled with an IncuCyte apoptosis reagent and
in PS asymmetry. NucLight live-cell nuclear labeling reagent to measure apoptotic cell death,
cell proliferation and kinetically monitor anti-proliferative effects of
• Assays are not amenable to long-term compounds.
measurements due to increasing signal
background over time. • Addition of IncuCyte apoptosis reagents to normal, healthy cells are non-
perturbing to cell growth or morphology and yield little to no intrinsic
• Manipulations can result in the loss of cells fluorescent signal.
or critical data in experiments where cells
undergo apoptosis at different rates according • IC50 and EC50 values can be calculated using kinetic area under the curve
to treatment conditions. values of nuclear counts and caspase-3/7 or annexin V counts, respectively.
• 96- and 384-well assay using a homogeneous “mix and read” protocol which
can be run over multiple days in full media. No wash or lifting steps required,
negating the concern that cells are lost during the experiment or labeling process.
• The assay has high statistical reproducibility and can be used both for single-
point screening or concentration response profiling.

• All data points and temporal data curves can be validated by individual
images or time-lapse movies respectively. The kinetic readout of the
IncuCyte® system provides both high definition (HD) phase as well as
quantitative fluorescent imaging.

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