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Apoptosis AssaysAssays and reagents to examine the structure, function, and physiology of apoptosis |
What is apoptosis?
Cell 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.
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].
Figure 1. Comparison of apoptosis and necrosis.
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 assays to detect cell apoptosis. Understanding the mechanism of cell death represents a critical aspect of toxicological profiling and plays a critical role in drug discovery efforts.
Interested in the non-apoptotic form of cell death? Learn more about Ferroptosis Research Solutions.
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.
Caspase enzyme activity assays
The activation of caspase enzymes occurs very early in the apoptotic process. These enzymes have been identified as crucial mediators of the complex biochemical events associated with apoptosis.
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.
Plasma membrane integrity and asymmetry assays
Changes in the cell’s membrane include distortion of the cell membrane, changes in the lipid composition and membrane integrity, and translocation of the phospholipid phosphatidylserine.
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).
- Ion indicators, including calcium, magnesium, pH, potassium and zinc
- Oxidative stress detection
Classic products for cell viability and autophagy can also be used to help characterize and identify cells undergoing apoptosis.
- Cell viability assays for flow cytometry, HCS, imaging and microplate readers
- Autophagy assays
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.
Table 2. Example of products for key apoptosis pathway proteins.
Learn more about the Cellular Apoptosis Pathway, Apoptosis Death Receptor Pathway and the CTL-Mediated Apoptosis Pathway
Related apoptosis assays and reagents
Citations:
Tools and handbooks
Fluorescence SpectraViewer
Supports all levels of experimental complexity, use the tool to compare excitation and emission spectra of fluorophores and reagents.
Flow cytometry protocols handbook
Protocols that fit your needs in flow cytometry ranging from sample preparation to numerous cell stimulation conditions, staining, immunophenotyping, and data analysis strategies.
Imaging protocol handbook
Protocols that fit your needs in imaging ranging from sample and assay preparation to staining, labeling, and data analysis strategies.
Resources
- Apoptosis Information in Cell Analysis Learning Center
Get application notes, webinars, and other useful education resources.
Molecular Probes Handbook
Support
Cell Analysis Support Center
Find technical information, tips and tricks, and answers to everyday problems.
For Research Use Only. Not for use in diagnostic procedures.
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