Estimation of Membrane Potential by Flow Cytometry

Howard M. Shapiro1

1 Howard M. Shapiro, M.D., P.C., West Newton, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.6
DOI:  10.1002/0471142956.cy0906s28
Online Posting Date:  May, 2004
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Abstract

In most living cells, differences between interior and exterior concentrations of ions (e.g., Na+, K+, Cl) generate an electrical potential across the cytoplasmic membrane. Membrane potential, like other cellular parameters, may change early in the course of surface receptor‐mediated cell activation processes related to the development, differentiated function, and pathology of a large number of cell types. Changes in ionic environment may play a role in transmembrane signaling in response to cell surface ligand‐receptor interactions. Thus, it is frequently of scientific interest to estimate membrane potential in individual cells. This unit provides methods for accomplishing this goal by flow or static cytometry using any of a number of cationic or anionic lipophilic fluorescent probes known as distributional dyes. The first of the two protocols is able to estimate the membrane potential of eukaryotic or bacterial cells. The second is more accurate, but can only be used with bacteria.

Keywords: membrane potential; ionic environment; ligand‐receptor interactions; fluorescent lipophilic dyes; mitochondrial membrane potential

     
 
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Table of Contents

  • Basic Protocol 1: Estimation of ΔΨ in Eukaryotic Cells or Bacteria
  • Basic Protocol 2: Measurement of ΔΨ in Bacteria Using DiOC2(3)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Estimation of ΔΨ in Eukaryotic Cells or Bacteria

  Materials
  • Cell culture
  • Hanks' balanced salt solution (HBSS) with calcium and magnesium but without phenol red (e.g., Life Technologies or appendix 2A)
  • Dimethyl sulfoxide (DMSO)
  • 1 mM gramicidin D (Sigma) in DMSO (store at 4°C to 25°C; stable at least 1 month)
  • 1 mM valinomycin (Sigma) in DMSO (store at 4°C to 25°C; stable at least 1 month)
  • Dye working solution (see recipe): 20 µM DiIC 1(3) or DiIC 1(5); or 10 µM DiOC 5(3) or DiOC 6(3); or 20 µM DiBAC 4(3) or DiSBAC 2(3)
  • 12 × 75–mm tubes
  • Additional reagents and equipment for cell culture and handling ( appendix 3B)

Basic Protocol 2: Measurement of ΔΨ in Bacteria Using DiOC2(3)

  Materials
  • Bacterial culture (typically 1 × 108 to 1 × 109 cells/ml)
  • Mueller‐Hinton broth (Life Technologies) with 50 mg/liter Ca2+ (MHBc)
  • Dimethyl sulfoxide (DMSO)
  • 2 mM valinomycin (Sigma) in DMSO (store at 4°C to 25°C; stable at least 1 month)
  • 2 mM CCCP (Sigma) in DMSO (store at 4°C; stable for at least 1 month)
  • Dye working solution: 3 mM DiOC 2(3) in DMSO (store at 4°C; stable for at least 1 month)
  • 12 × 75–mm tubes
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Figures

Videos

Literature Cited

Literature Cited
   Chen, L.B. 1989. Fluorescent labeling of mitochondria. Methods Cell Biol. 29:103‐123.
   Ehrenberg, B., Montana, V., Wei, M.D., Wuskell, J.P., and Loew, L.M. 1988. Membrane potential can be determined in individual cells from the Nernstian distribution of cationic dyes. Biophys. J. 53:785‐794.
   Gonzalez, J.E. and Tsien, R.Y. 1997. Improved indicators of cell membrane potential that use fluorescence resonance energy transfer. Chem. Biol. 4:269‐277.
   Jepras, R.I., Carter, J., Pearson, S.C., Paul, F.E., and Wilkinson, M.J. 1995. Development of a robust flow cytometric assay for determining numbers of viable bacteria. Appl. Environ. Microbiol. 61:2696‐2701.
   Kaprelyants, A.S. and Kell, D.B. 1992. Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry. J. Appl. Bacteriol. 72:410‐422.
   Kessel, D., Beck, W.T., Kukuruga, D., and Schulz, V. 1991. Characterization of multidrug resistance by fluorescent dyes. Cancer Res. 51:4665‐4670.
   López‐Amorós, R., Comas, J., and Vives‐Rego, J. 1995. Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation‐survival in seawater using rhodamine 123, propidium iodide, and oxonol. Appl. Environ. Microbiol. 61:2521‐2526.
   Mason, D.J., Allman, R., Stark, J.M., and Lloyd, D. 1994. Rapid estimation of bacterial antibiotic susceptibility with flow cytometry. J. Microsc. 176:8‐16.
   Mason, D.J., Power, E.G.M., Talsania, H., Phillips, I., and Gant, V.A. 1995. Antibacterial action of ciprofloxacin. Antimicrob. Agents Chemother. 39:2752‐2758.
   Montana, V., Farkas, D.L., and Loew, L.M. 1989. Dual‐wavelength ratiometric fluorescence measurements of membrane potential. Biochemistry 28:4536‐4539.
   Novo, D., Perlmutter, N.G., Hunt, R.H., and Shapiro, H.M. 1999. Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry 35:55‐63.
   Novo, D., Perlmutter, N.G., Hunt, R.H., and Shapiro, H.M. 2000. Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. Antimicrob. Agents Chemother. 44:827‐834.
   Ordóñez, J.V. and Wehman, N.M. 1993. Rapid flow cytometric antibiotic susceptibility assay for Staphylococcus aureus. Cytometry 14:811‐818.
   Robinson, J.P., Carter, W.O., and Narayanan, P.K. 1997. Functional assays by flow cytometry. In ASM Manual of Clinical Laboratory Immunology, 5th ed. (N.R. Rose, E.C. de Macario, J.P. Folds, H.C. Lane, and, R.M. Nakamura, eds.) pp. 245‐254. ASM Press, Washington, DC.
   Rottenberg, H. and Wu, S. 1998. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta 1404:393‐404.
   Shapiro, H.M. 1982. Cytological Assay Procedure. U.S. Patent No. 4,343,982, issued Aug. 10, 1982.
   Shapiro, H.M. 1988. Practical Flow Cytometry, 2nd ed., pp. 296‐298. Alan R. Liss, New York.
   Shapiro, H.M. 2000. Membrane potential estimation by flow cytometry. Methods 21:271‐276.
   Shapiro, H.M. 2003. Practical Flow Cytometry, 4th ed., pp. 385‐402, 519‐522. John Wiley & Sons, Hoboken, N.J.
   Shapiro, H.M., Natale, P.J., and Kamentsky, L.A. 1979. Estimation of membrane potentials of individual lymphocytes by flow cytometry. Proc. Natl. Acad. Sci. U.S.A. 76:5728‐5730.
   Silverman, J.A., Perlmutter, N.G., and Shapiro, H.M. 2003. Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob. Agents Chemother. 47:2538‐2544.
   Sims, P.J., Waggoner, A.S., Wang, C.H., and Hoffman, J.F. 1974. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 13:3315‐3330.
   Smiley, S.T., Reers, M., Mottola‐Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele, G.D. Jr., and Chen, L.B. 1991. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J‐aggregate‐forming lipophilic cation JC‐1. Proc. Natl. Acad. Sci. U.S.A. 88:3671‐3675.
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