Question:
What are the most commonly used Apoptosis Assays?
The Protein Man Says:
Apoptosis research necessitates the use of a wide range of apoptosis assays since the process itself is quite complicated. In addition, there is no single parameter that can define cell death in all systems so you should use several approaches in going about your research.
Main Types of Apoptosis Assays
There are five distinct types of apoptosis assays that are currently being used in apoptosis research. This includes caspase assays, DNA fragmentation assays, cell permeability assays and mitochondrial assays. In addition, there is also a type of assay that can be used to facilitate apoptosis signalling pathway analysis.
Caspase assays. The presence of caspase enzymes in any given sample is a sure indicator that complex biochemical events associated with apoptosis are taking place. As such, caspase assays can be used to detect apoptosis in cell populations during its earliest stages. These assays routinely use specific substrates that are digested by caspases to release colorimetric of fluorometric substrates.
DNA fragmentation assays. Apoptosis and DNA cleavage goes hand in hand so you can determine the extent of apoptosis by measuring the DNA fragments present in your sample. Most researchers use apoptopic DNA ladder analysis, ELISA quantification and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays for this purpose.
Cell permeability assays. Middle stage apoptosis can be determined by using cell permeability assays. As you probably know, healthy cells have rigid plasma membranes while dead cells have highly permeable cell membranes. With their highly compromised membranes, fluorescent dyes can easily pass through apoptopic cells. When this happens, you will observe a characteristic fluorescence or staining pattern that indicates the presence of apoptopic cell populations in your sample.
The increase in cell permeability also results in the loss of cytosolic proteins from the cell. Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme that is released into the culture medium following loss of membrane integrity resulting from either apoptosis or necrosis. LDH activity, therefore, can be used as an indicator of cell membrane integrity and serves as a general means to assess cytotoxicity resulting from chemical compounds or environmental toxic factors. Several assays exist for the quantification of released LDH.
Mitochondrial assays. Mitochondrial assays can help determine early stage apoptosis since it uses a cationic dye that gives off a different fluorescence when used in healthy cell populations and apoptopic cell populations.
Knowing which apoptosis assays to use and when to use them can greatly aid you in your research so you better start knowing them by heart.
