Page 30 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

Kinetic Caspase-3/7 and Annexin Apoptosis Assays

Quantitative assays for apoptotic pathway analysis
for both drug discovery and basic research

Introduction

Apoptosis, the biological process by which cells undergo The regulated loss of plasma membrane phosphatidylserine (PS)
programmed cell death, is required for normal tissue maintenance symmetry is also a classical marker of apoptosis. Dying cells trigger
and development. However, aberrations in apoptotic signaling the translocation of the normally inward-facing PS to the cellular
networks are implicated in numerous human diseases including surface, allowing for early phagocytic recognition of the dying cell
neurodegeneration and cancer. Apoptotic pathways are initiated by surrounding phagocytes.
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by extrinsic factors that result in activation of pro-apoptotic
receptors on the cell surface, or intrinsically by many different Numerous enzymatic, plate-reader and flow-cytometric assays
stimuli such as DNA damage, hypoxia, the absence of growth have been designed to measure caspase-3/7 activation or PS
factors, defective cell cycle control, or other types of cellular stress externalization. Most caspase-3/7 assays involve luciferase,
that result in release of cytochrome C from mitochondria. colorimetric or fluorometric reagent substrates that incorporate
a DEVD (Asp-Glu-Val-Asp) peptide motif which is recognized
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Stimulation of either the extrinsic or intrinsic apoptotic pathways by the enzyme. Annexin V is a recombinant protein with a high
triggers a signaling cascade that results in the activation of a family affinity and selectivity for PS residues, allowing it to be used
of proteins that play a major role in carrying out the apoptotic for the detection of apoptosis. Apoptosis assays using annexin V
process called caspases. Caspases (cysteinyl aspartate proteinases) conjugated to a fluoroprobe have been optimized for detection of
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cleave substrates following an Asp (D) amino acid residue. Effector PS externalization and are most commonly measured by flow-
targets of caspases include caspase family members themselves, cytometry.
proteins involved in fragmentation of cellular DNA (caspase-
activated DNAses), nuclear lamins, as well as proteins that make up In this chapter, we will examine kinetic approaches for measuring
the cell cytoskeleton. Caspase proteins are traditionally separated apoptosis using IncuCyte® Caspase-3/7 and Annexin V fluorescent
into two groups, initiator caspases (caspase 2, 8, 9 and 10), and reagents. Unlike plate reader and flow-cytometric endpoint
executioner or effector caspases (caspase 3, 6, and 7). As a primary approaches, kinetic live-cell image-based analysis allows for the
executioner caspase in most systems, the activation of caspase-3 evaluation of time-dependent effects of treatments or cell-
often results in the irreversible commitment of a cell to apoptosis. specific responses.
Therefore, the activation of caspase-3 is considered a reliable marker
for cells undergoing apoptosis.

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