Uncompensated Polychromatic Analysis of Mitochondrial Membrane Potential Using JC‐1 and Multilaser Excitation

Sara De Biasi1, Lara Gibellini1, Andrea Cossarizza1

1 Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Modena
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.32
DOI:  10.1002/0471142956.cy0732s72
Online Posting Date:  April, 2015
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Abstract

The lipophilic cation JC‐1 (5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzimidazolyl carbocyanine iodide) has been used for more than 20 years as a specific dye for measuring mitochondrial membrane potential (ΔΨm). In this unit, we revise our original protocol (that made use of a single 488 nm laser for the detection of monomers and aggregates, and where compensation was an important step) to use dual‐laser excitation. Moreover, thanks to recently developed multilaser instruments and novel probes for surface and intracellular markers, JC‐1 can be utilized by polychromatic flow cytometry to simultaneously detect, without any compensation between fluorescences, ΔΨm along with other biological parameters, such as apoptosis and the production of reactive oxygen species. © 2015 by John Wiley & Sons, Inc.

Keywords: apoptosis; mitochondrial membrane potential; JC‐1; polychromatic flow cytometry; Annexin V; CellRox

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Basic Determination of Mitochondrial Membrane Potential Using JC‐1: Dual‐Laser Excitation of the Dye Avoids Compensation Issues
  • Basic Protocol 2: Analysis of ΔΨm, Apoptosis, and Reactive Oxygen Species Content by 4‐Laser Polychromatic Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Basic Determination of Mitochondrial Membrane Potential Using JC‐1: Dual‐Laser Excitation of the Dye Avoids Compensation Issues

  Materials
  • Experimental samples: human peripheral blood lymphocytes or monocytes, or human tumor cell lines (e.g., RKO, HL60, U937, MCF7); here we use RKO cells, which derive from a colon carcinoma and grow adherent to the plastic flask
  • Complete RPMI culture medium, 1 ml per sample
  • 1 M valinomycin [dissolve valinomycin (mol. wt. 1111.32; Sigma‐Aldrich) 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 Aldrich)
  • 2.5 mg/ml JC‐1 (5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolylcarbocyanine iodide): prepare by dissolving JC‐1 (Life Technologies, Thermo Fisher Scientific) in dimethylformamide (DMF); store in a glass container up to 2 years at –20°C, protected from light
  • Phosphate‐buffered saline (PBS)
  • 3.5‐ml, 55 × 12–mm plastic tubes (Sarstedt, or equivalent)
  • Centrifuge (Minifuge RF; Heraeus), or equivalent
  • Flow cytometer equipped with a 488‐nm blue laser and with a 561‐nm yellow laser, e.g., Attune NxT (Life Technologies)
  • Additional reagents and equipment for counting ( appendix 3A) and culturing ( appendix 3B) mammalian cells

Basic Protocol 2: Analysis of ΔΨm, Apoptosis, and Reactive Oxygen Species Content by 4‐Laser Polychromatic Flow Cytometry

  Materials
  • Cells in culture (ATCC): in suspension or adherent in 24‐well tissue culture plate (as in protocol 1, we use RKO cells derived from human colon carcinoma
  • Complete RPMI culture medium
  • Phosphate‐buffered saline (PBS)
  • CellRox Deep Red Reagent (Life Technologies)
  • 2.5 mg/ml JC‐1 (5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolylcarbocyanine iodide); prepare by dissolving JC‐1 (Life Technologies, Thermo Fisher Scientific) in dimethylformamide (DMF); store in a glass container up to 2 years at –20°C, protected from light
  • Annexin V binding buffer (see recipe)
  • Pacific Blue‐conjugated annexin V (Life Technologies, Thermo Fisher Scientific): store at 4°C, protected from light
  • 3.5 ml, 55 × 12–mm plastic tubes (Sarstedt, or equivalent)
  • Centrifuge (Minifuge RF; Heraeus), or equivalent.
  • Attune NxT cytometer or equivalent cytometer equipped with four light sources for excitation at 405 nm (violet laser, for Annexin V), 488 and 561 nm (blue and yellow lasers, for JC‐1), and 638 nm (red laser, for CellRox) and filters for collecting fluorescence emissions at 455/40 (for annexin V), 520/20 (for JC‐1 monomers), 585/42 (JC‐1 aggregates), and 660/40 (CellRox)
  • Additional reagents and equipment for counting ( appendix 3A) and culturing ( appendix 3B) mammalian cells and detaching adherent cells using trypsin (see appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
  Abu, N., Akhtar, M.N., Yeap, S.K., Lim, K.L., Ho, W.Y., Zulfadli, A.J., Omar, A.R., Sulaiman, M.R., Abdullah, M.P., and Alitheen, N.B. 2014. Flavokawain a induces apoptosis in MCF‐7 and MDA‐MB231 and inhibits the metastatic process in vitro. PLoS One 6;9:e105244.
  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. 17:37‐49.
  Cossarizza, A. and Salvioli, S. 2000. Flow cytometric analysis of mitochondrial membrane potential Using JC‐1. Curr. Protoc. Cytom. 13:9.14.1‐9.14.7.
  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′tetrachloro1,1′,3,3′tetraethylbenzimidazol‐ carbocyanine iodide (JC‐1). Biochem. Biophys. Res. Commun. 197:40‐45.
  Cossarizza, A., Mussini, C., Mongiardo, N., Borghi, V., Sabbatini, A., De Rienzo, B., and Franceschi, C. 1997. Mitochondria alterations and dramatic tendency to undergo apoptosis in peripheral blood lymphocytes during acute HIV syndrome. AIDS 11:19‐26.
  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., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A., and Malorni, W. 1995. Protective effect of N‐acetylcysteine in tumor necrosis factor‐alpha‐induced apoptosis in U937 cells: The role of mitochondria. Exp. Cell Res. 220:232‐240.
  Cossarizza, A., Pinti, M., Moretti, L., Bricalli, D., Bianchi, R., Troiano, L., Fernandez, M.G., Balli, F., Brambilla, P., Mussini, C., and Viganò, A. 2002. Mitochondrial functionality and mitochondrial DNA content in lymphocytes of vertically infected human immunodeficiency virus‐positive children with highly active antiretroviral therapy‐related lipodystrophy. J. Infect. Dis. 185:299‐305.
  Galluzzi, L., Kepp, O., and Kroemer, G. 2012. Mitochondria: Master regulators of danger signalling. Nat. Rev. Mol. Cell Biol. 13:780‐788.
  Gibellini, L., De Biasi, S., Pinti, M., Nasi, M., Riccio, M., Carnevale, G., Cavallini, G.M., Sala de Oyanguren, F.J., O'Connor J.E, Mussini, C., De Pol, A., and Cossarizza, A. 2012. The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes. AIDS 26:2017‐2026.
  Green, D.R., Galluzzi, L., and Kroemer, G. 2011. Mitochondria and the autophagy‐inflammation‐cell death axis in organismal aging. Science 333:1109‐1112.
  Jelley, E.E. 1936. Spectral absorption and fluorescence of dyes in the molecular state. Nature 138:1009‐1010.
  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 275:1132‐1136.
  Kroemer, G., Galluzzi L., and Brenner, C. 2007. Mitochondrial membrane permeabilization in cell death. Physiol. Rev. 87:99‐163.
  Lizarbe, M.A., Barrasa, J.I., Olmo, N., Gavilanes, F., and Turnay, J. 2013. Annexin‐phospholipid interactions. Functional implications. Int. J. Mol. Sci. 14:2652‐2683.
  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 71:334‐344.
  Lugli, E., Roederer, M., and Cossarizza, A. 2010. Data analysis in flow cytometry: The future just started. Cytometry A 77:705‐713.
  Marringa, W.J., Krueger, M.J., Burritt, N.L., and Burritt, J.B. 2014. Honey bee hemocyte profiling by flow cytometry. PLoS One. 9:e108486.
  Martel, C., Wang, Z., and Brenner, C. 2014. VDAC phosphorylation, a lipid sensor influencing the cell fate. Mitochondrion.
  Perelman, A., Wachtel, C., Cohen, M., Haupt, S., Shapiro, H., and Tzur, A. 2012. JC‐1: Alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 3:e430.
  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 69:1162‐1173.
  Polla, B.S., Kantengwa, S., François, D., Salvioli, S., Franceschi, C., Marsac, C., and Cossarizza A. 1996. Mitochondria are selective targets for the protective effects of heat shock against oxidative injury. Proc. Natl. Acad. Sci. U.S.A. 93:6458‐6643.
  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., Nozzi, J.L., and Nason, M.C. 2011. SPICE: Exploration and analysis of post‐cytometric complex multivariate datasets. Cytometry A 79:167‐174.
  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 delta psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411:77‐82.
  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 40:189‐197.
  Troiano, L., Ferraresi, R., Lugli, E., Nemes, E., Roat, E., Nasi, M., Pinti, M., and Cossarizza, A. 2007. Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nat. Protoc. 2:2719‐2727.
  Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssiere, J.L., Petit, P.X., and Kroemer, G. 1995. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181:1661‐1672.
Internet Resources
  https://www.lifetechnologies.com/it/en/home/references/molecular‐probes‐the‐handbook.html?icid=fr‐handbooks‐1
  Source for The Handbook—A Guide to Fluorescent Probes and Labeling Technologies from Invitrogen/Molecular Probes.
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