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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Abstract

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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Materials

Basic Protocol 1:

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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   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.
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   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.
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