Abstract
Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.
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References
Wyllie, A.H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556 (1980).
Susin, S.A. et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J. Exp. Med. 184, 1331–1341 (1996).
Ferlini, C. et al. Sequence of metabolic changes during X-ray-induced apoptosis. Exp. Cell Res. 247, 160–167 (1999).
Ferlini, C. et al. Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques. Cytometry 24, 106–115 (1996).
Darzynkiewicz, Z., Staiano-Coico, L. & Melamed, M.R. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. USA 78, 2383–2387 (1981).
Ferlini, C., Biselli, R., Nisini, R. & Fattorossi, A. Rhodamine 123: a useful probe for monitoring T cell activation. Cytometry 21, 284–293 (1995).
Petit, J.M., Maftah, A., Ratinaud, M.H. & Julien, R. 10N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria. Eur. J Biochem. 209, 267–273 (1992).
Garcia Fernandez, M.I., Ceccarelli, D. & Muscatello, U. Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models. Anal. Biochem. 328, 174–180 (2004).
Belikova, N.A. et al. Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes. Biochemistry 45, 4998–5009 (2006).
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Ferlini, C., Scambia, G. Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange. Nat Protoc 2, 3111–3114 (2007). https://doi.org/10.1038/nprot.2007.397
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DOI: https://doi.org/10.1038/nprot.2007.397
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