Flow Cytometric Analysis of Mitochondrial Membrane Potential Using JC‐1

Andrea Cossarizza1, Stefano Salvioli1

1 University of Modena, Modena, Italy
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.14
DOI:  10.1002/0471142956.cy0914s13
Online Posting Date:  May, 2001
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Changes in membrane potential have long been known to represent early activation events. This unit presents very recent developments in both fluorescent probes and functional applications and demonstrates the use of the JC‐1 probe for measuring mitochondrial membrane potential by flow cytometry. A valuable component of this measurement system is the possibility of making quantitative measurements of changes in membrane potential.

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Cells in suspension (e.g., human peripheral blood lymphocytes, monocytes, or human tumor cell lines such as HL‐60, U937, K562; appendix 3B)
  • Complete cell culture medium (e.g., complete RPMI with 10% FBS; appendix 2A)
  • Depolarizing drug: e.g., 0.1 mM valinomycin in DMSO or 0.25 mM carbonyl cyanide m‐(trifluoromethoxy)phenylhydrazone (FCCP) in DMSO
  • 1 mg/ml JC‐1 stock solution in dimethyl sulfoxide (DMSO); store in small aliquots at −20°C for up to 1 year
  • PBS ( appendix 2A)
  • Flow cytometer equipped with a 488‐nm excitation light source and band‐pass filters centered around 525 and 590 nm
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Literature Cited

Literature Cited
   Collins, J.M. and Foster, K.A. 1983. Differentiation of promyelocytic (HL‐60) cells into mature granulocytes: Mitochondrial‐specific rhodamine 123 fluorescence. J. Cell Biol. 96:94‐99.
   Cossarizza, A., Baccarani, Contri M., Kalashnikova, G., and Franceschi, C. 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J‐aggregate forming lipophilic cation 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolcarbocyanine iodide (JC‐1). Biochem. Biophys. Res. Commun. 197:40‐45.
   Cossarizza, A., Kalashnikova, G., Grassilli, E., Chiappelli, F., Salvioli, S., Capri, M., Barbieri, D., Troiano, L., Monti, D., and Franceschi, C. 1994. Mitochondrial modifications during rat thymocyte apoptosis: A study at the single cell level. Exp. Cell Res. 214:323‐330.
   Cossarizza, A., Salvioli, S., Franceschini, M.G., Kalashnikova, G., Barbieri, D., Monti, D., Grassilli, E., Tropea, F., Troiano, L., and Franceschi, C. 1995. Mitochondria and apoptosis: A cytofluorimetric approach. Fund. Clin. Immunol. 3:67‐68.
   Cossarizza, A., Ceccarelli, D., and Masini, A. 1996. Functional heterogeneity of isolated mitochondrial population revealed by cytofluorimetric analysis at the single organelle level. Exp. Cell Res. 222:84‐94.
   Cossarizza, A., Mussini, C., Borghi, V., Mongiardo, N., Nuzzo, C., Pedrazzi, J., Benatti, F., Moretti, L., Pinti, M., Franceschi, C., and De Rienzo, B. 1999. Apoptotic features of peripheral blood mononuclear granulocytes and monocytes during primary, acute HIV infection. Exp. Cell Res. 247:304‐311.
   Darzynkiewicz, Z., Staiano‐Coico, L., and Melamed, M.R. 1981. Increased mitochondrial uptake of rhodamine 123 during lymphocyte stimulation. Proc. Natl. Acad. Sci. U.S.A. 78:2383‐2387.
   Goldstein, S. and Korczack, L.B. 1981. Status of mitochondria in living human fibroblasts during growth and senescence in vitro: Use of the laser dye rhodamine 123. J. Cell Biol. 91:392‐398.
   Hada, H., Honda, C., and Tanemura, H. 1977. Spectroscopic study on the J‐aggregate of cyanine dyes. I. Spectral changes of UV bands concerned with J‐aggregate formation. Photogr. Sci. Eng. 21:83‐91.
   Jenssen, H.‐L., Redmann, K., and Mix, E. 1986. Flow cytometric estimation of transmembrane potential of macrophages—A comparison with microelectrode measurements. Cytometry 7:339‐346.
   Johnson, L.V., Walsh, M.L., and Chen, L.B. 1980. Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. U.S.A. 77:990‐994.
   Kroemer, G., Zamzani, N., and Susin, S.A. 1997. Mitochondrial control of apoptosis. Immunol. Today 18:44‐51.
   Lopez‐Mediavilla, C., Orfao, A., Gonzales, M., and Medina, J.M. 1989. Identification by flow cytometry of two distinct rhodamine‐123‐stained mitochondrial populations in rat liver. FEBS Lett. 254:115‐120.
   Maftah, A., Petit, J.M., Ratinaud, M.H., and Julien, R. 1989. 10‐N‐Nonyl‐acridine orange: A fluorescent probe which stains mitochondria independently of their energetic state. Biochem. Biophys. Res. Commun. 164:185‐190.
   Nadakavukaren, K.K., Nadakavukaren, J.J., and Chen, L.B. 1985. Incresased rhodamine 123 uptake by carcinoma cells. Cancer Res. 45:6093‐6099.
   Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., and Gougeon, M.‐L. 1995. Alterations in mitochondrial structure and function are early events of dexamethasone‐induced thymocyte apoptosis. J. Cell Biol. 130:157‐167.
   Reers, M., Smith, T.W., and Chen, L.B. 1991. J‐aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30:4480‐4486.
   Richter, C., Schweizer, M., Cossarizza, A., and Franceschi, C. 1996. Control of apoptosis by the cellular ATP level. FEBS Lett. 378:107‐110.
   Salvioli, S., Ardizzoni, A., Franceschi, C., and Cossarizza, A. 1997. JC‐1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess ΔΨ in intact cells. Implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411:77‐82.
   Skulachev, V.P. 1996. Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide‐producing mitochondria and cell. FEBS Lett. 397:7‐10.
   Smiley, S.T., Reers, M., Mottola‐Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele, G.D., and Chen, L.B. 1991. Intracellular heterogeneity in mitochondrial membrane potential revealed by a J‐aggregate‐forming lipophilic cation JC‐1. Proc. Natl. Acad. Sci. U.S.A. 88:3671‐3675.
   Terasaki, M., Song, J., Wong, J.R., Weiss, M.J., and Chen, B.L. 1984. Localization of endoplasmic reticulum in living and glutaraldehyde‐fixed cells with fluorescent dyes. Cell 38:101‐108.
   Tiso, M., Gangemi, R., Bargellesi‐Severi, A., Pizzolitto, S., Fabbi, M., and Risso, A. 1995. Spontaneous apoptosis in human thymocytes. Am. J. Pathol. 147:434‐444.
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