Measurement of Mitochondrial Membrane Potential with the Fluorescent Dye Tetramethylrhodamine Methyl Ester (TMRM)

  • Sarah Creed
  • Matthew McKenzieEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1928)


The mitochondrial membrane potential (Δψm) drives the generation of ATP by mitochondria. Interestingly, Δψm is higher in many cancer cells comparted to healthy noncancerous cell types, providing a unique metabolic marker. This feature has also been exploited for therapeutic use by utilizing drugs that specifically accumulate in the mitochondria of cancer cells with high Δψm. As such, the assessment of Δψm can provide very useful information as to the metabolic state of a cancer cell, as well as its potential for malignancy. In addition, the measurement of Δψm can also be used to test the ability of novel anticancer therapies to disrupt mitochondrial metabolism and cause cell death.

Here, we outline two methods for assessing Δψm in cancer cells using confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester (TMRM). In the first protocol, we describe a technique to quantitatively measure Δψm, which can be used to compare Δψm between different cell types. In the second protocol, we describe a technique for assessing changes to Δψm over time, which can be used to determine the effectiveness of different therapeutic compounds or drugs in modulating mitochondrial function.

Key words

Mitochondria Membrane potential TMRM Cancer cells Osteosarcoma Confocal imaging Fluorescence 



This work was supported by the Monash Health Translational Precinct (MHTP) Micro Imaging Platform and the Victorian Government’s Operational Infrastructure Support Program.


  1. 1.
    Wallace DC (1999) Mitochondrial diseases in man and mouse. Science 283:1482–1488CrossRefGoogle Scholar
  2. 2.
    Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA (2011) Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques 50:98–115CrossRefGoogle Scholar
  3. 3.
    Chinopoulos C, Tretter L, Adam-Vizi V (1999) Depolarization of in situ mitochondria due to hydrogen peroxide-induced oxidative stress in nerve terminals: inhibition of alpha-ketoglutarate dehydrogenase. J Neurochem 73:220–228CrossRefGoogle Scholar
  4. 4.
    Rottenberg H, Wu S (1998) Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim Biophys Acta 1404:393–404CrossRefGoogle Scholar
  5. 5.
    McKenzie M, Liolitsa D, Akinshina N, Campanella M, Sisodiya S, Hargreaves I, Nirmalananthan N, Sweeney MG, Abou-Sleiman PM, Wood NW, Hanna MG, Duchen MR (2007) Mitochondrial ND5 Gene Variation Associated with Encephalomyopathy and Mitochondrial ATP Consumption. J Biol Chem 282:36845–36852CrossRefGoogle Scholar
  6. 6.
    McKenzie M, Duchen MR (2016) Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids. PLoS One 11:e0154371CrossRefGoogle Scholar
  7. 7.
    Zhang X, Lemasters JJ (2013) Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes. Free Radic Biol Med 63:243–253CrossRefGoogle Scholar
  8. 8.
    Zhang BB, Wang DG, Guo FF, Xuan C (2015) Mitochondrial membrane potential and reactive oxygen species in cancer stem cells. Fam Cancer 14:19–23CrossRefGoogle Scholar
  9. 9.
    Ye XQ, Li Q, Wang GH, Sun FF, Huang GJ, Bian XW, Yu SC, Qian GS (2011) Mitochondrial and energy metabolism-related properties as novel indicators of lung cancer stem cells. Int J Cancer 129:820–831CrossRefGoogle Scholar
  10. 10.
    Bonnet S, Archer SL, Allalunis-Turner J, Haromy A, Beaulieu C, Thompson R, Lee CT, Lopaschuk GD, Puttagunta L, Harry G, Hashimoto K, Porter CJ, Andrade MA, Thebaud B, Michelakis ED (2007) A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 11:37–51CrossRefGoogle Scholar
  11. 11.
    Lamb R, Ozsvari B, Bonuccelli G, Smith DL, Pestell RG, Martinez-Outschoorn UE, Clarke RB, Sotgia F, Lisanti MP (2015) Dissecting tumor metabolic heterogeneity: Telomerase and large cell size metabolically define a sub-population of stem-like, mitochondrial-rich, cancer cells. Oncotarget 6:21892–21905PubMedPubMedCentralGoogle Scholar
  12. 12.
    Britten CD, Rowinsky EK, Baker SD, Weiss GR, Smith L, Stephenson J, Rothenberg M, Smetzer L, Cramer J, Collins W, Von Hoff DD, Eckhardt SG (2000) A phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clin Cancer Res 6:42–49PubMedGoogle Scholar
  13. 13.
    Lo-Coco F, Avvisati G, Vignetti M, Thiede C, Orlando SM, Iacobelli S, Ferrara F, Fazi P, Cicconi L, Di Bona E, Specchia G, Sica S, Divona M, Levis A, Fiedler W, Cerqui E, Breccia M, Fioritoni G, Salih HR, Cazzola M, Melillo L, Carella AM, Brandts CH, Morra E, von Lilienfeld-Toal M, Hertenstein B, Wattad M, Lubbert M, Hanel M, Schmitz N, Link H, Kropp MG, Rambaldi A, La Nasa G, Luppi M, Ciceri F, Finizio O, Venditti A, Fabbiano F, Dohner K, Sauer M, Ganser A, Amadori S, Mandelli F, Dohner H, Ehninger G, Schlenk RF, Platzbecker U (2013) Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med 369:111–121CrossRefGoogle Scholar
  14. 14.
    Lim SC, Carey KT, McKenzie M (2015) Anti-Cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH:ubiquinone oxidoreductase (Complex I). Am J Cancer Res 5:689–701PubMedPubMedCentralGoogle Scholar
  15. 15.
    Duchen MR, Surin A, Jacobson J (2003) Imaging mitochondrial function in intact cells. Methods Enzymol 361:353–389CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Monash Micro ImagingHudson Institute of Medical ResearchClaytonAustralia
  2. 2.Centre for Innate Immunity and Infectious DiseasesHudson Institute of Medical ResearchClaytonAustralia
  3. 3.Department of Molecular and Translational ScienceMonash UniversityClaytonAustralia
  4. 4.School of Life and Environmental SciencesDeakin UniversityWaurn PondsAustralia

Personalised recommendations