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Mitochondrial Bioenergetics pp 103–117Cite as

Safranine as a Fluorescent Probe for the Evaluation of Mitochondrial Membrane Potential in Isolated Organelles and Permeabilized Cells

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 810))

Abstract

The mitochondrial electrical membrane potential (Δψ) is the main component of the proton motive force (Δp) generated across the inner mitochondrial membrane during electron flow through the respiratory chain. Among the techniques available to assess Δψ, methods that rely on the spectrophotofluorometric responses of dyes are widely employed for whole suspensions of isolated mitochondria or permeabilized cells. Safranine is one of the dyes currently used most often for this purpose. Safranine is a lipophilic cationic dye that undergoes optical shifts upon its potential-dependent distribution between the external medium and the intramitochondrial compartment and on its stacking to inner mitochondrial membrane anionic sites. The association between the optical changes of safranine and the membrane potential allows unknown Δψ values to be estimated from an equation describing their relationship. Here, we describe the use of safranine as a fluorescent indicator of Δψ in isolated mitochondria and digitonin-permeabilized cells. We present suitable conditions to employ safranine as a Δψ indicator.

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Acknowledgments

The authors are currently supported by grants from the following Brazilian agencies: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Instituto Nacional de Obesidade e Diabetes. T.R.F. and D.R.M. are graduate students supported by FAPESP fellowships.

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Authors and Affiliations

  1. Faculdade de Ciências Médicas, Departamento de Patologia Clínica, Universidade Estadual de Campinas, Campinas, SP, 13083-887, Brazil

    Tiago R. Figueira & Roger F. Castilho

  2. Faculdade de Ciências Médicas, Departamento de Patologia Clínica, Universidade Estadual de Campinas, Rua Dr Quirino, 1856 – Centro, Campinas, SP, 13083-887, Brazil

    Daniela R. Melo

  3. Faculdade de Ciências Médicas, Departamento de Patologia Clínica, Universidade Estadual de Campinas, 177 Rua Roxo Moreira, Campinas, SP, 13083-887, Brazil

    Aníbal E. Vercesi

Authors
  1. Tiago R. Figueira
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  2. Daniela R. Melo
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  3. Aníbal E. Vercesi
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  4. Roger F. Castilho
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Corresponding author

Correspondence to Roger F. Castilho .

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Editors and Affiliations

  1. Center for Neurosciences and Cell Biolog, Universidad de Coimbra, Lg. Marquês de Pombal 3046, Coimbra, 3004-517, Portugal

    Carlos M. Palmeira

  2. Centro de Neurociencias, Universidad de Coimbra, Lg. Marquês de Pombal 3046, Coimbra, 3004-517, Portugal

    António J. Moreno

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Figueira, T.R., Melo, D.R., Vercesi, A.E., Castilho, R.F. (2012). Safranine as a Fluorescent Probe for the Evaluation of Mitochondrial Membrane Potential in Isolated Organelles and Permeabilized Cells. In: Palmeira, C., Moreno, A. (eds) Mitochondrial Bioenergetics. Methods in Molecular Biology, vol 810. Humana Press. https://doi.org/10.1007/978-1-61779-382-0_7

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  • DOI: https://doi.org/10.1007/978-1-61779-382-0_7

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-381-3

  • Online ISBN: 978-1-61779-382-0

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