Drawing of apoptotic cell with apoptotic bodies

What is apoptosis?

Apoptosis, also known as programmed cell death, is a highly regulated process that not only allows for proper growth and development by ridding the organism of unneeded cells and tissues, but also minimizes threats to the organism by destroying surplus cells of the immune system and virus-infected or DNA-damaged cells [1].

Programmed cell death is morphologically and biochemically distinct from cell death by injury (necrosis). Unlike necrotic cells, apoptotic cells exhibit compaction of the nuclear chromatin, shrinkage of the cytoplasm, and production of membrane-bound apoptotic bodies, as well as DNA fragmentation and cleavage or degradation of several cellular proteins (Figure 1). Biochemically, apoptosis is distinguished by fragmentation of the genome and cleavage or degradation of several cellular proteins. Incorrectly regulated apoptosis is implicated in a number of disease states, including cancer, stroke, Alzheimer’s disease, and several autoimmune diseases.

Illustration of the differences in cellular processes of a cell undergoing apoptosis vs necrosis

Figure 1. Comparison of apoptosis and necrosis.

Apoptosis can be induced through three different pathways: 1) targeting mitochondria functionality (mitochondrial, cellular or apoptosis intrinsic pathway), 2) direct transduction of the signal via adaptor proteins (death-receptor or apoptosis extrinsic pathway) and 3) the perforin/granzyme pathway [2-3].

Identifying cells that are going through apoptosis can be challenging because many of the assays used as indicators are detecting structural and functional changes that occur in other processes as well. Additionally, no single parameter fully defines programmed cell death in all systems and the appearance of these changes can vary with apoptotic pathway or cell types. Therefore, it is often advantageous to use several different approaches when studying apoptosis.

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Apoptosis assays for cell structure, function and physiology

Mitochondria function and membrane potential assays

A distinctive feature of apoptosis is the disruption of active mitochondria, which includes changes in the membrane potential and alterations to the oxidation–reduction potential of the mitochondria. These changes are thought to be due to the opening of the mitochondrial permeability transition pore, allowing passage of ions and small molecules as well as cytochrome c release.

Caspase enzyme activity assays

The activation of caspase enzymes occurs very early in the apoptotic process. These enzymes comprise a family of cysteine–aspartic acid specific proteases that have been identified as crucial mediators of the complex biochemical events associated with apoptosis. Also see the section below for antibodies and other tools to detect caspases.

DNA fragmentation and morphological assays

The characteristic breakdown of the nucleus during apoptosis includes collapse and fragmentation of the DNA, degradation of the nuclear envelope and nuclear blebbing, resulting in the formation of micronuclei. We offer multiple types of assays for detection of these morphological changes.

Plasma membrane integrity and asymmetry assays

Changes in the cell’s membrane can be seen throughout the apoptosis process. Distortion or blebbing of the cell membrane begins early and as apoptosis progresses, changes in the lipid composition and in membrane integrity become more prevalent. One such change is the translocation of the phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane, exposing phosphatidylserine to the external cellular environment.

Additional reagents to monitor apoptotic cell physiology

Changes in the cell cytosol also occur during apoptosis, with variations in cellular ion concentration, pH, and level of reactive oxygen species (ROS).

Classic products for cell viability and autophagy can also be used to help characterize and identify cells undergoing apoptosis.

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Tools for analyzing apoptosis pathways

Apoptosis is a highly regulated process and involves a significant number of signaling pathways. Thermo Fisher Scientific offers a wide range of primary antibodies, ELISA kits, multiplexed immunoassays, peptides and recombinant proteins as well as genomic assays targeted to specific proteins. Below is just an example of the types of assays we have available for some key apoptosis proteins.

Learn more about the Cellular Apoptosis Pathway, Apoptosis Death Receptor Pathway and the CTL-Mediated Apoptosis Pathway

Table 2. Example of products for key apoptosis pathway proteins.

Protein Full name Primary antibodies ELISA Kits Proteins/peptides TaqMan assays
AIF Apoptosis Inducing Factor AIF antibodies   AIF proteins/peptides AIF TaqMan Assays
Apaf-1 Apoptotic protease activating factor Apaf-1 antibodies Apaf-1 ELISAs   Apaf-1 TaqMan Assays
Bcl Protein Family
(Bcl-2, Bcl-10, Bcl-x, Bcl-XL, Bcl-XS, Bcl-w)
B-cell lymphoma protein Bcl Protein Family antibodies Bcl Protein Family ELISAs Bcl Protein Family proteins/peptides Bcl Protein Family TaqMan Assays
t-BID/BID-p15 Truncated BH3-interacting domain death agonist;  BH3-interacting domain death agonist p15 t-BID/BID-p15 antibodies     t-BID/BID-p15 TaqMan Assays
Caspase-3 Cysteinyl aspartic acid-protease-3 Caspase-3 antibodies Caspase-3 ELISAs Caspase-3 proteins/peptides Caspase-3 TaqMan Assays
Caspase-7 Cysteinyl aspartic acid-protease-7 Caspase-7 antibodies   Caspase-7 proteins/peptides Caspase-7 TaqMan Assays
Caspase-9 Cysteinyl aspartic acid-protease-9 Caspase-9 antibodies Caspase-9 ELISAs Caspase-9 proteins/peptides Caspase-9 TaqMan Assays
Cytochrome c   Cytochrome c antibodies Cytochrome c ELISAs   Cytochrome c TaqMan Assays
Fas/CD95   Fas/CD95 antibodies Fas/CD95 ELISAs Fas/CD95 proteins/peptides Fas/CD95 TaqMan Assays
Fas-ligand/CD178   Fas-ligand/CD178 antibodies Fas-ligand/CD178 ELISAs   Fas-ligand/CD178 TaqMan Assays
Smac/DIABLO Second mitochondrial activator of caspases/direct Smac/DIABLO antibodies Smac/DIABLO ELISAs Smac/DIABLO proteins/peptides Smac/DIABLO TaqMan Assays

Find more apoptosis-related assays and reagents

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Resources

Apoptosis Pathways
Apoptosis is the tightly regulated process of controlled cell death in multicellular organisms. Learn more about several of the important pathways.

Molecular Probes Handbook

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