( Apoptosis ...)Apoptosis detection by annexin V and,,,active caspase-3 with the Agilent 2100,,,bioanalyzer

Apoptosis
Apoptosis, or programmed cell death, is a genetically controlled response of cells to commit suicide. The purpose of this process is to kill unwanted host cells. It is used in three situations: for development and homeostasis, as a defense mechanism and in aging. Apoptosis is characterized by a distinct set of morphological events involving plasma membrane blebbing, loss of cell volume, nuclear condensation, fragmentation of DNA at nucleosomal intervals and ultimate fragmentation of the cell into membraneenclosed apoptotic bodies1.
During the early phase of apoptosis, changes occur at the plasma membrane. Phosphatidyl serine (PS) that is actively confined to the inner leaflet of the lipid bilayer in healthy cells translocates to the outer layer, where it is exposed at the external surface of the cell2. Annexin V belongs to the family of calcium and phospholipid binding proteins with high affinity for PS3. It can be used as a sensitive probe for PS exposure on the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, as it also appears during necrosis. These mechanisms of cell death differ in their initial stages when the cell membrane remains intact during apoptosis but loses integrity, leaking cellular contents, during necrosis4. Therefore, measuring annexin V binding to the cell surface as indicator for apoptosis has to be performed in conjunction with a calcein retention test that verifies the integrity of the cell membrane. As electrically neutral or nearly neutral molecules, calcein acetyloxymethyl (AM) esters freely diffuse into most cells. Once inside the cell, these non-fluorescent substrates are converted by nonspecific intracellular esterases into fluorescent products that are retained by cells with intact plasma membranes. In contra
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