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Apoptosis is a form of cell death characterized by several features including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering, and the eventual engulfment of the cell by phagosomes. You can assay apoptosis using a number of different approaches.
During apoptosis, the caspases (cysteine-aspartate proteases) accelerate cell death through the proteolysis of over 400 proteins. Caspases are activated through the intrinsic and extrinsic cell death pathways.
The intrinsic cell death pathway is governed by the Bcl-2 family of proteins, which regulate commitment to cell death through the mitochondria. The key step in the intrinsic cell death pathway is the permeabilization of the mitochondrial outer membrane, after which cells are committed to cell death. Following permeabilization, the release of proteins from the mitochondrial intermembrane space promotes caspase activation and apoptosis. Released cytochrome C binds APAF-1, inducing the activation of caspase 9. Caspase 9 then activates caspases 3 and 7, leading to apoptosis.
Activation of the extrinsic cell death pathway occurs following the binding on the cell surface of “death receptors” to their corresponding ligands such as Fas, TNFR1 or TRAIL. These death receptors recruit adaptor molecules such as FADD and caspase 8, which then activate caspase 3 and caspase 7, leading to apoptosis.
Apoptosis occurs via a complex signaling cascade. The image below shows the main parameters of apoptosis and the approximate relative time when markers for those events are likely to be detected.
These parameters do not happen in a sequential order, and many of them will overlap and occur at the same time.
As cell death can occur by several different paths, including apoptosis, necrosis, autophagy, and necroptosis, some of which share characteristics, you need to examine multiple apoptosis markers to confirm that this is the mechanism of cell death in your experimental system.
The table below shows the main apoptosis markers and the most common methods to study them.
Flow cytometry analysis of annexin V binding
Cleavage of anti-apoptotic Bcl-2 family proteins
Western blot assessment of protein cleavage
Colorimetric / fluorometric substrate-based assays in microtiter plates
Detection of cleavage of the fluorometric substrate in flow cytometry/microscopy or by microtiter plates analysis
Western blot analysis of pro- and active caspase
Flow cytometry/microscopy analysis with antibodies specifically recognizing the active form of caspases
Microplate spectrophotometry analysis with antibodies specifically recognizing the active form of caspases
Caspase substrate (PARP) cleavage
Microplate spectrophotometry analysis with antibodies specific for cleaved PARP
Western blot analysis of cleaved PARP
Colorimetric/fluorometric substrate-based assays in microtiter plates
Flow cytometry/ microscopy/microplate spectrophotometry analysis with Δψm sensitive probes
Oxygen consumption studies
Western blot analysis of the presence of cytochrome C in the cytosol
Antibody-based microscopy analysis of the presence of cytochrome C in the cytosol
Flow cytometry analysis of sub G1 peak
Flow cytometry analysis of chromatin condensation
Microscopy analysis of chromatin condensation
Analysis of DNA ladder in agarose gel
Analysis of DNA fragmentation by TUNEL
Light microscopy analysis of membrane blebbing
Western blot analysis of cleaved substrate (gelsolin, ROCK1)
There are a number of methods for running an apoptosis assay to measure these markers of apoptosis.
Other assay methods are used to assay necrosis, anoikis and autophagy.
Annexin V binds to phosphatidylserine, which migrates to the outer plasma membrane in apoptosis. Analysis is typically by flow cytometry. Pair Annexin V with a membrane impermeable dye like 7-AAD to distinguish between intact, apoptotic, and necrotic cells (eg see ab214663, ab214484, or ab214485).
Activated caspases can be detected using antibodies with IHC, western blotting, or flow cytometry.
Caspase activity assays either use peptide substrates, which are cleaved by caspases in cell extracts, or similar substrates that bind to activated caspases in live cells. Caspase specificity varies by substrate.
Learn more about our assays for caspases 1 through 12, formulated either for cell lysates with analysis by plate reader or for live cells with analysis by flow cytometer, microscope or plate reader.
Dyes that accumulate in mitochondria due to the mitochondrial membrane potential are also used in the analysis of apoptosis. For more information, see our guide to cell viability assays. Apoptotic cells stain more weakly with these dyes due to the loss of membrane potential.
Cytochrome C is released into the cytoplasm following total loss of mitochondrial membrane potential.
Western blot, fluorescence microscope
The glutathione assay is also used for the analysis of apoptosis.
Fluorometric plate reader