Flow Cytometric Methods of Analyzing Apoptotic Cells

Apoptosis is a physiological, programmed mode of cell death, which is necessary for tissue modeling and organogenesis in embryonic development and in the control of homeostasis in a diversity of tissue types (1 ). The distinct morphological features of apoptosis clearly distinguish it from the passive mode of cell death, necrosis, which is an unprogrammed response to toxic stimuli. The diagnosis of apoptosis relies on detection of these morphological changes, i.e., condensation and fragmentation of chromatin, cell shrinkage associated with cytoplasmic condensation, and retention of cell membrane integrity. These changes may be accompanied by the fragmentation of the cell into membrane-bound apoptotic bodies containing cytoplasmic organelles, nuclear components, or both (2 ). Classical methods of diagnosing apoptosis include light and electron microscopy in which the above morphological changes can be visualized, and internucleosomal DNA fragmentation assays in which the fragmentation of DNA can be seen after gel electrophoresis as a ladder pattern representing the generation of multiple oligonucleosomal fragments. The widespread use of in vitro systems for studying cell death has led many researchers to turn to flow cytometry as an alternative to the classic methods of analyzing apoptosis, since it offers the advantages of rapid analysis of individual cells, low cell number requirement, and the opportunity for simultaneous measurement of several cellular parameters.