Abstract
Apoptosis is a genetically regulated process by which cells can be eliminated in vivo in response to a wide range of physiological and toxicological signals. Cells in vitro may be induced to die by apoptosis, e.g., by depletion of nutrients or survival factors from the culture media. Described here are several biochemical and cytometric techniques of varying complexity that have been developed to detect cellular and subcellular changes occurring during apoptosis. For all procedures, cells can be obtained from either monolayer or suspension culture.
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References
Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257.
Al-Rubeai, M. (1998) Apoptosis and cell culture technology, in Advances in Biochemical Engineering/Biotechnology (Scheper, T., ed.), Springer-Verlag. New York, pp. 225–249.
Ishaque, A. and Al-Rubeai, M. (1997) Detection and analysis of apoptosis by flow cytometry, in Protocols in Cell and Tissue Culture (Griffiths, J. B., Doyle, A., and Newell, D. G., eds.), John Wiley, New York.
Al-Rubeai, M. and Singh, R. (1998) Apoptosis in cell culture. Curr. Opin. Biotechnol. 9, 152–156.
Singh, R. and Al-Rubeai, M. (1998) Analysis of apoptosis by fluorescence microscopy, in Protocols in Cell and Tissue Culture, Techniques for Cell Characterization and Analysis (Griffiths, J. B., Doyle, A., and Newell, D. G., eds.), John Wiley, New York.
Ishaque, A. and Al-Rubeai, M. (1999) Role of Ca, Mg and K ions in determining apoptosis and extent of suppression afforded by bcl-2 during hybridoma cell culture. Apoptosis 4/5, 335–355.
Ishaqe, A. and Al-Rubeai, M. (2002) Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture. Apoptosis 7, 231–239.
Ishaque, A. and Al-Rubeai, M. (2004) Monitoring of apoptosis, in Cell Engineering IV. Kluwer Academic Publishers, Dordrecht.
Al-Rubeai M. and Emery A. N. (1993) How cytometry in animal cell culture. Bio/technology 11, 573–579.
Dive, C., Gregory, C. D., Phipps, D. J., Evans, D. L., Milner, A. E., and Wyllie, A. H. (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiplarameter flow cytometry. Biochim. Biophys. Acta 1133, 275–285.
Darzynkiewicz, Z., Robinson, J. P., and Crissman, H. A. (1994) Assays of cell viability: discrimination of cells dying by apoptosis. Methods Cell Biol. 41, 15–38.
Nicoletti, I, Migiliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C. (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–280.
Darzynkiewicz, Z., Bruno, S., Del Bino, G., et al. (1992). Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795–808.
Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. (1997) Cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry 27, 1–20.
Ormerod, M. G., Sun, X. M., Snowden, R. T., Davies, R., Fearnhead, H., and Cohen, G. M. (1993) Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. Cytometry 14, 595–602.
Koopman, G., Reutlingsperger, C. P. M., and Kuijten, G. A. M. (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415.
Collins, M. K., Marvel, J., Malde, P., and Lopez-Rivas, A. (1992) Interleukin 3 protects murine bone marrow cells from apoptosis induced by DNA damaging agents. J. Exp. Med. 176, 1043–1051.
Elstein, K. H. and Zucker, R. M. (1994) Comparison of cellular and nuclear flow cytometric techniques for discriminating apoptotic sub-populations. Exp. Cell Res. 211, 322–331.
Klucar, J. and Al-Rubeai, M. (1997) G2 cell cycle arrest and apoptosis are induced in Burkitt’s lymphoma cells by the anticancer agent oracin. FEBS Lett. 400, 127–130.
Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207.
Ishaque, A. and Al-Rubeai, M. (1998) Use of intracellular pH and annexin V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture J. Immunol. Methods 221, 43–57.
Gorczyca, W., Bruno, S., and Darzynkiewicz, Z. (1992) DNA strand breaks occuring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int. J. Oncol. 1, 639–648.
Gold, R., Schmeid, M., and Rothe, G. (1993) Detection of DNA fragmentation in apoptosis-application of in situ nick translation to cell culture systems and tissue-sections. J. Histochem. Cytochem. 41, 1023–1030.
Green, D. R. and Reed, J. C. (1998) Mitochondria and apoptosis. Science 281, 1309–1313.
Thornberry, N. A. and Lazebnik. (1998) Caspases: enemies within. Science 281, 1312–1316.
Ishaque, A., Sorokin, A., and Dunn, M. J. (2001) Cyclooxygenase-2 inhibits TNF-alpha mediated apoptosis in renal glomerular mesangial cells. J. Biol. Chem. 278(12), 10,629–10,640.
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Ishaque, A., Al-Rubeai, M. (2007). Measurement of Apoptosis in Cell Culture. In: Pörtner, R. (eds) Animal Cell Biotechnology. Methods in Biotechnology, vol 24. Humana Press. https://doi.org/10.1007/978-1-59745-399-8_13
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DOI: https://doi.org/10.1007/978-1-59745-399-8_13
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