The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL‐60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only Sphase HL‐60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: (a) Activation of an endonuclease in apoptotic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA‐specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity oaf the apoptotic process. (b) Plasma membrane integrity, which is lost in necrotic but not in apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early‐ and late‐apoptotic cells. (c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. (d) The ATP‐dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. (e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. (f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. (g) The decrease in forward light shatter, paralleled either by no change (HL‐60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. (h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at love pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. (i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis. The data presented indicate that flow cytometry can be applied in basic research on molecular and biochemical mechanisms of apoptosis, as well as in the clinic, where the ability to monitor early signs of apoptosis in samples from patients' tumors may be predictive of the outcome of some treatment protocols. © 1992 Wiley‐Liss, Inc.

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology.

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The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology. Cytometry 27:1–20, 1997. © 1997 Wiley‐Liss, Inc.

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The impact of complement on cancer metastasis has not been well studied. In this report, we demonstrate in a preclinical mouse model of breast cancer that the complement anaphylatoxin C5a receptor (C5aR)