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Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells

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Part of the Methods in Molecular Biology book series (MIMB,volume 2497)

Abstract

The mitochondrial membrane potential (ΔψM) is the major component of the bioenergetic driving force responsible for most cellular ATP produced, and it controls a host of biological processes. In intact cells, assay readouts with commonly used fluorescence ΔψM probes are distorted by factors other than ΔψM. Here, we describe a protocol to calculate both ΔψM and plasma membrane potential (ΔψP) in absolute millivolts in intact single cells, or in populations of adherent, cultured cells. Our approach generates unbiased data that allows comparison of ΔψM between cell types with different geometry and ΔψP, and to follow ΔψM in time when ΔψP fluctuates. The experimental paradigm results in fluorescence microscopy time courses using a pair of cationic and anionic probes with internal calibration points that are subsequently computationally converted to millivolts on an absolute scale. The assay is compatible with wide field, confocal or two-photon microscopy. The method given here is optimized for a multiplexed, partial 96-well microplate format to record ΔψP and ΔψM responses for three consecutive treatment additions.

Key words

  • Mitochondrial membrane potential
  • Plasma membrane potential
  • Mitochondrial biogenesis
  • Fluorescence microscopy
  • Live cell microscopy
  • Cell culture
  • Tetramethylrhodamine methyl ester
  • TMRM
  • Bis-oxonol
  • MitoTracker
  • Cellular heterogeneity
  • Single cell

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  • DOI: 10.1007/978-1-0716-2309-1_2
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Acknowledgments

The authors thank Kareem Heslop (Hollings Cancer Center, Medical University of South Carolina, SC. USA) for critical reading of the manuscript, Christopher Wiley and Leyla Teos (Buck Institute for Research on Aging, CA, USA) for providing cultures of BJ1 cells and freshly isolated cardiomyocytes, respectively. This work was supported by the National Institutes of Health (NIH) grants 1R41DA043369 to AAG and R01AG055822 to Simon Melov. AAG declares a financial interest in Image Analyst Software.

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Correspondence to Akos A. Gerencser .

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Lerner, C.A., Gerencser, A.A. (2022). Unbiased Millivolts Assay of Mitochondrial Membrane Potential in Intact Cells. In: Tomar, N. (eds) Mitochondria. Methods in Molecular Biology, vol 2497. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2309-1_2

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  • DOI: https://doi.org/10.1007/978-1-0716-2309-1_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2308-4

  • Online ISBN: 978-1-0716-2309-1

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