Apoptosis assays and markers guide
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An introduction to the mechanisms of apoptotic cell death and apoptosis assays and markers.
Apoptosis is a form of programmed cell death that has important roles in development, aging, and disease. Apoptosis is initiated by a tightly regulated signaling cascade that results in caspase activation. Several features characterize apoptosis, including cell shrinkage, membrane blebbing, chromosome condensation, nuclear fragmentation, DNA laddering, and the eventual engulfment of the cell by phagosomes.
Overview:
Introduction to apoptosis mechanisms
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.
Learn more about the mechanisms of apoptosis and other forms of cell death in our three comprehensive guides to apoptosis, necroptosis, and autophagy.
Apoptosis marker guide
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 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.
Apoptosis marker | Detection methods |
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 Δ ψm 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) |
Apoptosis assay kits guide
There are a number of methods for running an apoptosis assay to measure these markers of apoptosis.
- Annexin V binding of cell surface phosphotidylserine
- DNA condensation and fragmentation (TUNEL) assays
- Caspase activation and detection assays
- Mitochondrial membrane potential-dependent dyes
- Cytochrome C release assays
- Glutathione assays
Other assay methods are used to assay necrosis, anoikis and autophagy.
Apoptosis pathway knockout cell lines panel
To further investigate the up and downstream signaling pathways relevant to apoptosis we offer a panel of CRISPR engineered KO cell lines. These panels include a collection of six knockout cell lines of genes involved in the apoptotic cell death pathway, all provided in one kit for your convenience, with matching parental cell lines as controls.
Annexin V assay
Annexin V binds to phosphatidylserine, which migrates to the outer plasma membrane in apoptosis. The analysis is typically done 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).
See a partial list of our Annexin V dye conjugates below or a full list here.
DNA condensation/fragmentation
DNA condensation in apoptosis can be measured using DNA stains to visualize condensed nuclei.
DNA fragmentation can be measured using agarose gels. In the TUNEL assay (view the TUNEL staining / TUNEL Assay guide), the 3’ ends of DNA fragments are labeled with deoxyuridine either conjugated to a fluorescent dye or biotin.
Active caspase detection
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.
We also offer assays for cathepsin and calpain activity analysis: cathepsin D (ab65302), cathepsin B (ab65303), cathepsin L (ab65306), and calpains (ab65308).
Mitochondrial membrane potential-dependent dyes
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 release
Cytochrome C is released into the cytoplasm following total loss of mitochondrial membrane potential.
Assay | Instrument | Assay kits |
Cytochrome C | Western blot, fluorescence microscope |
Glutathione assay
The glutathione assay is also used for the analysis of apoptosis.
Assay | Instrument | Assay kits |
GSH/GSSG assay | Fluorometric plate reader |
Necrosis, anoikis, and autophagy
We offer several kits for studying other forms of cell death: necrosis and apoptosis (ab176749 and ab176750), anoikis (ab211153), and autophagy (ab133075 and ab139484).