Polychromatic Analysis of Mitochondrial Membrane Potential Using JC‐1

Enrico Lugli1, Leonarda Troiano1, Andrea Cossarizza1

1 Department of Biomedical Sciences, General Pathology Section, University of Modena and Reggio Emilia, Modena, null
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.32
DOI:  10.1002/0471142956.cy0732s41
Online Posting Date:  July, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Dissipation of the mitochondrial membrane potential (m) has been accepted as a hallmark of some apoptotic processes. Depending on the model studied, it can occur during the early stages of cell death or later on, after loss of DNA integrity. Discordant data in the literature could be the consequence of using improper probes such as rhodamine 123 (R123) or DiOC6(3), which are not always appropriate for the study of m. The lipophilic cation JC-1, a specific probe for measuring m, is currently the gold standard. Thanks to recently developed instruments and additional probes for cell surface and intracellular markers, it can be used in polychromatic flow cytometric assays to simultaneously detect m along with other biological parameters.

Keywords: apoptosis; mitochondrial membrane potential; JC-1; polychromatic flow cytometry; Hoechst 33342; Annexin V; propidium iodide

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Basic Determination of Mitochondrial Membrane Potential Using JC-1
  • Alternate Protocol: Analysis of m with Mitotracker RED CMXRos
  • Basic Protocol 2: Analysis of m Together with Several Apoptotic Parameters by Polychromatic Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Basic Determination of Mitochondrial Membrane Potential Using JC-1

 Materials
  • Experimental samples: human peripheral blood lymphocytes or monocytes, or human tumor cell lines (e.g., HL60, U937, K562)
  • Complete RPMI culture medium (see appendix 3B), 1 ml per sample
  • 1 mM valinomycin: dissolve valinomycin (mol. wt. 1111.32; Sigma) in dimethylformamide (DMF) and store in a glass container up to 6 months at 4°C or
  • 1 mM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP; Sigma)
  • 1.25 mg/ml JC-1 (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide): prepare by dissolving JC-1 (Invitrogen) in dimethylformamide (DMF); store in a glass container up to 2 years at –20°C, protected from light
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 3.5-ml, 55×12–mm plastic tubes (Sarstedt, or equivalent)
  • Centrifuge (Minifuge RF; Heraeus), or equivalent
  • 20-, 200-, and 1000-µl micropipettors (e.g., Pipetman; Gilson)
  • Vortexer (e.g., Vibrofix VF1; Janke & Kunkel-Ika Labortechnik)
  • Flow cytometer equipped with a 488-nm blue laser, e.g., CyFlow (Partec) or FACScan (BD)
  • Additional reagents and equipment for counting (appendix 3A) and culturing (appendix 3B) mammalian cells

Alternate Protocol: Analysis of m with Mitotracker RED CMXRos

 Additional Materials (also see Basic Protocol 1)
  • Experimental cells in culture (e.g., HL60 promyelocytic cells; ATCC)
  • 1 mM staurosporine (Sigma)
  • 10 mM 10-nonyl bromide acridine orange (NAO): prepare by dissolving NAO (mol. wt. 472.51; Invitrogen) in methanol; store up to 1 year at –20°C, protected from light.
  • 1 mM MitoTracker Red CMXRos: prepare by dissolving MitoTracker Red CMXRos (mol. wt. 631.52; Invitrogen) in DMSO; store up to 1 year at –20°C, protected from light

Basic Protocol 2: Analysis of m Together with Several Apoptotic Parameters by Polychromatic Flow Cytometry

 Materials
  • Cells in culture (ATCC): in suspension or adherent in 24-well tissue culture plate
  • Complete RPMI culture medium (see appendix 3B)
  • 1 mg/ml Hoechst 33342: prepare by dissolving Hoechst 33342 (Invitrogen) in ethanol; store up to 6 months at –20°C, protected from light.
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 1.25 mg/ml JC-1 (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide): prepare by dissolving JC-1 (Invitrogen) in dimethylformamide (DMF): store in a glass container up to 2 years at –20°C, protected from light
  • Annexin V binding buffer (see recipe)
  • Alexa 647–conjugated Annexin V (Invitrogen): store at 4°C, protected from light
  • 10 µg/ml propidium iodide (PI): prepare by adding PI (1 mg/ml in water; Sigma) to PBS to a final concentration of 10 µg/ml; store up to 2 yr at 4°C, protected from light
  • 3.5-ml, 55×12–mm plastic tubes (Sarstedt, or equivalent)
  • Centrifuge (Minifuge RF; Heraeus), or equivalent
  • 20-, 200-, and 1000-µl micropipettors (e.g., Pipetman; Gilson)
  • Vortexer (e.g., Vibrofix VF1; Janke & Kunkel-Ika Labortechnik)
  • CyFlow ML (Partec), FACSCanto II (BD), or equivalent cytometer equipped with three light sources for excitation at 350 nm or 405 nm (UV or violet laser for Hoechst), 488 nm (blue laser for JC-1 and PI), and 633 nm (red laser for Alexa 647) and filters for collecting fluorescence emissions at 455/40 (Hoechst), 520/20 (JC-1 monomers), 575/25 (JC-1 aggregates), 625/40 (PI), and 660/40 (Alexa 647)
  • Additional reagents and equipment for counting (appendix 3A) and culturing (appendix 3B) mammalian cells and detaching adherent cells using trypsin (see appendix 3B)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 7.32.1 Effect on the fluorescence of the dye JC-1 due to mitochondrial membrane depolarization of HL-60 cells treated with valinomycin. (A) The control cells were untreated and stained with 2.5 µg/ml JC-1. (B) Note the shift to the bottom and to the right of cells with mitochondria depolarized by treatment with 100 nM valinomycin and stained with 2.5 µg/ml JC-1.
    View Image
  •  FigureFigure 7.32.2 Polychromatic analysis of apoptosis. U937 cells were cultured overnight in the absence (panel A) or presence (panel B) of 100 µM quercetin (to induce apoptosis) and then stained for polychromatic analysis of apoptosis, as described in Basic Protocol 2. Viable, apoptotic, and necrotic cells were identified by the combination of Annexin V and PI fluorescence, and m and DNA content were analyzed by JC-1 and Hoechst fluorescence, respectively, in these subsets. Note that apoptotic cells can be grouped into different populations according to high (h), intermediate (i), and low (l) m (separated by the bars in the central columns of both panels A and B) and different DNA content (normal or hypodiploid as in the right columns of both panels).
     FigureFigure 7.32.2(continued) Polychromatic analysis of apoptosis. U937 cells were cultured overnight in the absence (panel A) or presence (panel B) of 100 µM quercetin (to induce apoptosis) and then stained for polychromatic analysis of apoptosis, as described in Basic Protocol 2. Viable, apoptotic, and necrotic cells were identified by the combination of Annexin V and PI fluorescence, and m and DNA content were analyzed by JC-1 and Hoechst fluorescence, respectively, in these subsets. Note that apoptotic cells can be grouped into different populations according to high (h), intermediate (i), and low (l) m (separated by the bars in the central columns of both panels A and B) and different DNA content (normal or hypodiploid as in the right columns of both panels).
    View Image

Videos

Literature Cited

Literature Cited
    Bossy-Wetzel, E., Newmeyer, D.D., and Green, D.R. 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 1:37-49.
    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. 1:323-330.
    Jenssen, H.L., Redmann, K., and Mix, E. 1986. Flow cytometric estimation of transmembrane potential of macrophages–a comparison with microelectrode measurements. Cytometry 4:339-346.
    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. 2: 990-994.
    Kluck, R.M., Bossy-Wetzel, E., Green, D.R., and Newmeyer, D.D. 1997. The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 5303:1132-1136.
    Koopman, G., Reutelingsperger, C.P., Kuijten, G.A., Keehnen, R.M., Pals, S.T., and van Oers, M.H. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 5:1415-1420.
    Kroemer, G., Galluzzi, L., and Brenner, C. 2007. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 1:99-163.
    Lopez-Mediavilla, C., Orfao, A., Gonzalez, M., and Medina, J.M. 1989. Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver. FEBS Lett. 1-2:115-120.
    Lugli, E., Troiano, L., Ferraresi, R., Roat, E., Prada, N., Nasi, M., Pinti, M., Cooper, E.L., and Cossarizza, A. 2005. Characterization of cells with different mitochondrial membrane potential during apoptosis. Cytometry A 1:28-35.
    Lugli, E., Pinti, M., Nasi, M., Troiano, L., Ferraresi, R., Mussi, C., Salvioli, G., Patsekin, V., Robinson, J.P., Durante, C., Cocchi, M., and Cossarizza, A. 2007. Subject classification obtained by cluster analysis and principal component analysis applied to flow cytometric data. Cytometry A 5:334-344.
    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. 1:185-190.
    Mazzini, G., Ferrari, C., and Erba, E. 2003. Dual excitation multi-fluorescence flow cytometry for detailed analyses of viability and apoptotic cell transition. Eur. J. Histochem. 4:289-298.
    Petit, P.X., O'Connor, J.E., Grunwald, D., and Brown, S.C. 1990. Analysis of the membrane potential of rat- and mouse-liver mitochondria by flow cytometry and possible applications. Eur. J. Biochem. 2:389-397.
    Petrausch, U., Haley, D., Miller, W., Floyd, K., Urba, W.J., and Walker, E. 2006. Polychromatic flow cytometry: A rapid method for the reduction and analysis of complex multiparameter data. Cytometry A 12: 1162-1173.
    Poot, M., Zhang, Y.Z., Kramer, J.A., Wells, K.S., Jones, L.J., Hanzel, D.K., Lugade, A.G., Singer, V.L., and Haugland, R.P. 1996. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 12: 1363-1372.
    Poot, M., Gibson, L.L., and Singer, V.L. 1997. Detection of apoptosis in live cells by MitoTracker red CMXRos and SYTO dye flow cytometry. Cytometry 4:358-364.
    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 18:4480-4486.
    Roederer, M., Moore, W., Treister, A., Hardy, R.R., and Herzenberg, L.A. 2001. Probability binning comparison: A metric for quantitating multivariate distribution differences. Cytometry 1:47-55.
    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 changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 1:77-82.
    Salvioli, S., Dobrucki, J., Moretti, L., Troiano, L., Fernandez, M.G., Pinti, M., Pedrazzi, J., Franceschi, C., and Cossarizza, A. 2000. Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: Analysis at the single cell and single organelle level. Cytometry 3: 189-197.
    Terasaki, M., Song, J., Wong, J.R., Weiss, M.J., and Chen, L.B. 1984. Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell 1:101-108.
    Troiano, L., Granata, A.R., Cossarizza, A., Kalashnikova, G., Bianchi, R., Pini, G., Tropea, F., Carani, C., and Franceschi, C. 1998. Mitochondrial membrane potential and DNA stainability in human sperm cells: A flow cytometry analysis with implications for male infertility. Exp. Cell Res. 2:384-393.
    Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S.A., Petit, P.X., Mignotte, B., and Kroemer, G. 1995. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 2:367-377.
 Internet Resources
    http://probes.invitrogen.com/handbook

Source for The Handbook — A Guide to Fluorescent Probes and Labeling Technologies from Invitrogen/Molecular Probes.

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library