Quantitative Analysis of Apoptosis and Necrosis in Live Cells Using Flow Cytometry.

ABSTRACT

Apoptosis and necrosis are the two sides of the cell death penumbra. Apoptosis is a well-studied model of cell death wherein the cell destroys itself employing a predefined form of active signaling without the release of soluble cytoplasmic contents to the external environment. Compared to apoptosis, necrosis is a nonspecific form of sudden cell death in response to an invasive external stimulus which in turn is devoid of active programmed intracellular signaling leading to the sudden release of the soluble cellular contents consequent to the rupture of the cell membrane. This fundamental difference between apoptosis and necrosis made us believe that the former is the safe form of cell death and the latter is an undesirable one which often elicits an inflammatory response to the adjacent cells. Recent studies have shown that necrosis also involves a few defined cellular and complex biochemical events similar to apoptosis rendering it difficult to distinguish these two events at the single-cell level using the currently used popular assays.Here we provide a newly described detailed methodology encompassing cell system development along with a multiparametric flow cytometry-based approach to discriminate apoptotic cells from necrotic cells using a stable cell line expressing genetically encoded probe for detecting caspase activation and DsRed targeted at the mitochondria.