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Apoptosis  (Apoptosis and Programmed Cell death)

As cells become damaged or are no longer needed, they undergo apoptosis, or programmed cell death, a normal physiological process that occurs during embryonic development and tissue homeostasis. Apoptosis is an organized process that signals cells to self- destruct for cell renewal or to control aberrant cell growth. Apoptosis controls the orderly death of damaged cells, whereas necrosis occurs as a result of tissue damage, causing the loss of both damaged and surrounding cells. The apoptotic process is characterized by certain morphological features. These include changes in the plasma membrane (such as loss of membrane symmetry and loss of membrane attachment), a condensation of the cytoplasm and nucleus, protein cleavage, and inter-nucleosomal cleavage of DNA. In the final stages of the process, dying cells become fragmented into “apoptotic bodies” and consequently are eliminated by phagocytic cells without significant inflammatory damage to surrounding cells. However, some cell types do not display characteristic features of apoptosis. In those cases, multiple aspects of apoptosis might need to be analyzed to confirm the mechanism of cell death.

Annexin V: A Key Protein in Apoptosis Signaling. Changes in the plasma membrane are one of the first characteristics of the apoptotic process detected in living cells. Apoptosis can be detected by the presence of phosphatidylserine (PS), which is normally located on the cytoplasmic face of the plasma membrane. During apoptosis, PS translocates to the outer leaflet of the plasma membrane and can be detected by flow cytometry through binding to fluorochrome-labeled Annexin V when calcium is present. Since intracellular Annexin V is also exposed if the plasma membrane is compromised, a membrane-impermeant dye such as 7-AAD is commonly used to distinguish between apoptotic and excluded dead cells. The populations of cells that are stained with only Annexin V represent the apoptotic cell populations.

There are many apoptotic triggers including certain cytokines, protein-protein interactions, and chemicals.

Once apoptosis starts, changes in the mitochondria membrane potential can be measured by flow cytometry using the membrane permeable dye, JC-1. Increases in mitochondrial membrane potential lead to increased mitochondrial membrane permeability and the release of soluble proteins such as cytochrome c and pro-caspases.

Caspases are a series of proteases activated upon cleavage at aspartate residues during the earliest stages of apoptosis.  Active caspases can then cleave many proteins including Poly-ADP ribose polymerase (PARP) and other caspases. DNA fragmentation is one of the last phases in apoptosis resulting from the activation of endonucleases during the apoptotic process. There are several established methods for the study of DNA fragmentation. Flow cytometry can be used for analysis of the terminal deoxynucleotidyltransferase (TdT) nick end labeling (TUNEL method) to support the study of DNA fragmentation.

At the end of apoptosis, cells become completely non- viable as they lose membrane integrity and become permeable to membrane-impermeant dyes such as 7-AAD or fixable viability stains. These dyes allow the identification of dead cells for further analysis or for exclusion in multicolor panels where dead cells may non-specifically bind antibody, affecting experimental results. In the case of fixable viability stains, these reagents bind covalently to surface and intracellular amines, providing compatibility with protocols requiring fixation and permeabilization.

In addition to caspases and Annexin V, there are several other proteins important for the study of apoptosis, including the Bcl-2 family, tumor necrosis factor receptor (TNFR) family, PARP, and other signaling molecules. Bcl-2 family members, identified by the presence of conserved BCL2 homology (BH3) domains, are versatile key regulators of apoptosis. Bcl-2, for example, protects cells from apoptosis by associating with the mitochondrial membrane and preventing the release of cytochrome c from the mitochondria. In contrast, other Bcl-2family members such as Bax promote apoptosis. Increased levels of Bcl-2 have been reported in cancer.

Over the years, multicolor flow cytometry has become essential in the study of apoptosis. Success of the technology results from its ability to monitor these processes along with other cellular events, such as protein phosphorylation or cytokine secretion, within heterogeneous cell populations.

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