Advertisement

Mitochondria pp 421-430 | Cite as

Assessing Mitochondrial Potential, Calcium, and Redox State in Isolated Mammalian Cells Using Confocal Microscopy

  • Sean M. Davidson
  • Derek Yellon
  • Michael R. Duchen
Part of the Methods in Molecular Biology™ book series (MIMB, volume 372)

Abstract

Mitochondria play a vital role in the regulation of intracellular calcium dynamics. Fluorescent dyes can be used to provide a direct measurement of the redox state, mitochondrial membrane potential, and mitochondrial calcium content. The simplicity of this approach lends itself to high-throughput assays and time-resolved analyses; however, care must be taken to avoid artifactual results. We outline general methods using confocal microscopy for analysis of the redox state, mitochondrial membrane potential, and mitochondrial calcium content in adult cardiomyocytes. We demonstrate how these parameters can be analyzed in parallel using the emission spectra “fingerprinting” method even when emission spectra overlap.

Key Words

Calcium cardiomyocytes confocal microscopy membrane potential NADH redox state 

References

  1. 1.
    Duchen, M. R. (2000) Mitochondria and calcium: from cell signalling to cell death. J. Physiol. 529(Pt. 1), 57–68.CrossRefPubMedGoogle Scholar
  2. 2.
    Bernardi, P., Scorrano, L., Colonna, R., Petronilli, V., and Di Lisa, F. (1999) Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur. J. Biochem. 264, 687–701.CrossRefPubMedGoogle Scholar
  3. 3.
    Nicholls, D. G. and Ward, M. W. (2000) Mitochondrial membrane potential and neuronal glutamate excitotoxicity: mortality and millivolts. Trends Neurosci. 23, 166–174.CrossRefPubMedGoogle Scholar
  4. 4.
    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.CrossRefPubMedGoogle Scholar
  5. 5.
    Duchen, M. R., Surin, A., and Jacobson, J. (2003) Imaging mitochondrial function in intact cells. Methods Enzymol. 361, 353–389.CrossRefPubMedGoogle Scholar
  6. 6.
    Voronina, S. G., Barrow, S. L., Gerasimenko, O. V., Petersen, O. H., and Tepikin, A. V. (2004) Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating DeltaPsim. J. Biol. Chem. 279, 27,327–27,338.CrossRefPubMedGoogle Scholar
  7. 7.
    Chance, B., Schoener, B., Oshino, R., Itshak, F., and Nakase, Y. (1979) Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J. Biol. Chem. 254, 4764–4771.PubMedGoogle Scholar
  8. 8.
    Minta, A., Kao, J. P., and Tsien, R. Y. (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178.PubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Sean M. Davidson
    • 1
  • Derek Yellon
    • 1
  • Michael R. Duchen
    • 2
  1. 1.The Hatter Institute and Centre for CardiologyUniversity College LondonLondonUK
  2. 2.Department of PhysiologyUniversity College LondonLondonUK

Personalised recommendations