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 S-phase 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 apoptocic 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 of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not 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 scatter, 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 low 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.(ABSTRACT TRUNCATED AT 400 WORDS)

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.990130802

Features of apoptotic cells measured by flow cytometry

https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.1939-4640.2002.tb02599.x

Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques.

http://cau.ac.kr/~jjang14/BioMEMS/Ivnitski_BSBE_Bacteria_Detection_Bio_Sensors_Review_1999.pdf

Biosensors for detection of pathogenic bacteria

http://dbkgroup.org/Papers/davey_kell_micrev96.pdf

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Flow cytometry offers important advantages for the study of tumour cell biology. In particular, this powerful technique allows the identification of cell subpopulations within heterogeneous cell samples and permits simultaneous measurement and correlation of multiple cellular parameters. The explosion in flow cytometric analysis since 1979 when the first edition of 'Flow Cytometry and Sorting' was published, is reflected by the sheer size and weight and the small print used in this long awaited Second Edition! It is clearly not intended as 'an easy to follow' introduction to Flow Cytometry, however all established flow cytometry laboratories should certainly replace their now far outdated first edition. In order to follow Flow Cytometry and Sorting in its entirity the reader would need a working knowledge of at least physics, engineering, and computer science, together with fluorescence techniques and biochemistry and immunology! This edition is ambitious, comprising 39 chapters written by 70 authors expert in their area of flow cytometry. The chapters are divided into eight parts. Each chapter has been for the most part rewritten, and there are several additional

Flow Cytometry and Sorting

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Flow Cytometry and Sorting