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Apoptosis assay and marker guide

Your options when you need the best apoptosis assay or apoptosis marker, and an introduction to the mechanisms of apoptosis.

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.

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.

Cell death parameters

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.

Apoptosis marker

Detection methods

Loss of membrane asymmetry/ PS exposure

Flow cytometry analysis of annexin V binding

Cleavage of anti-apoptotic Bcl-2 family proteins

Western blot assessment of protein cleavage

Caspase activation

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

Non-caspase proteases (cathepsins and calpain) activation

Colorimetric/fluorometric substrate-based assays in microtiter plates

Mitochondrial transmembrane potential (Δ​ψm) decrease

Flow cytometry/ microscopy/microplate spectrophotometry analysis with Δ​ψm sensitive probes

Oxygen consumption studies

Cytochrome C release

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

Increase of sub G1 population

Flow cytometry analysis of sub G1 peak

Nuclear condensation

Flow cytometry analysis of chromatin condensation

Microscopy analysis of chromatin condensation

DNA fragmentation

Analysis of DNA ladder in agarose gel

Analysis of DNA fragmentation by TUNEL

Membrane blebbing

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.

Other assay methods are used to assay necrosis, anoikis and autophagy.

Annexin V assay

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).

See a partial list of our Annexin V dye conjugates below or a full list here.

Annexin V conjugate

Ex/Em

Assay kits

FITC

495/519

ab14085, ab14082

Cy3

548/561

ab14142, ab14143

Cy5

647/665

ab14150, ab14147

PE

496/576

ab14155, ab14154

PE-Cy5

565/693

ab14159

EGFP

488/530

ab14153, ab14152

Biotin


ab14190, ab14165


Cell health apoptosis fig 1


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, the 3’ ends of DNA fragments are labeled with deoxyuridine either conjugated to a fluorescent dye or biotin.

Assay

Instrument

Assay kits

DNA fragmentation

Gel electrophoresis

ab66090, ab65627, 
ab66093

TUNEL assay

Flow cytometry, fluorescence microscope, microscope

ab66108, ab66110, 
ab206386

Cell health apoptosis fig 2

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

ab110415, ab110417,
ab65311

Glutathione assay

The glutathione assay is also used for the analysis of apoptosis.

Assay

Instrument

Assay kits

GSH/GSSG assay

Fluorometric plate reader

ab138881


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).


Learn more about apoptosis here.


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