Identifying the Stages of Apoptosis
Apoptosis represents a highly regulated multibiochemical event. This pathway plays a key role in cellular development, like Hematopoiesis, and differs from necrosis in that apoptosis is controlled cell death.
Classification of the different types of cell death involves:
- Definition of the cellular structure
- Structural changes
- Membrane alteration
- Cellular processes changes
- Nuclear degradation
Apoptosis can be seen as a stage-dependent process from its induction to early, intermediate and late-stage apoptotic events. Methods and reagents have been developed to identify the various apoptotic stages, and to clearly distinguish them from necrotic processes.
Early Stage Apoptosis Identification
Early stage apoptosis is represented by changes to, and ultimate loss of, the mitochondrial membrane potential. Mitochrondrial membrane potential is detected by JC-1, a dye that selectively enters the mitochondria. In functional mitochondria, JC-1 forms aggregates which show a fluorescent emission at ~590 nm. With reduced membrane potential the dye forms monomers where the emission is shifted to ~530nm. This shift is used to measure mitochondrial membrane potential, and the entry of the cell into early stage apoptosis.
Early/Mid Stage Apoptosis Identification
Another key apoptotic process is the translocation of phosphatidylserin (PS), a protein located in the cellular membrane. PS in live, intact cells is cytosolic but translocates to the extracellular portion of the membrane in early/mid apoptosis. Annexin V can be used for PS detection where cell impermeable dyes, such as Propidium Iodide or 7-AAD, need to be used as a counterstain to differentiate early apoptosis from late apoptosis or necrosis.
Late Stage Apoptosis Identification
You can identify late stage apoptosis by looking at the defragmentation of DNA. Apo-Direct assay utilizes a method to detect the multitude of 3'-hydroxyl ends created during late stage apoptosis. Measurement is provided by a two color stain using fluorescein-deoxyuridine triphosphate (FITC-dUTP) for DNA Break detection and Propidium Iodide for total DNA counterstain.
And these are just a few ways to identify apoptosis….
Additional ways of Apoptosis/Necrosis detection are looking at metabolic changes, membrane changes, Caspases, Phosphoproteins, cellular calcium levels and many more.