Advertisement

The Journal of Physiological Sciences

, Volume 67, Issue 6, pp 731–737 | Cite as

Simple and inexpensive technique for measuring oxygen consumption rate in adherent cultured cells

  • Eiji Takahashi
  • Yoshihisa Yamaoka
Technical Note

Abstract

Measurement of cellular oxygen consumption rate (OCR) is essential in assessing roles of mitochondria in physiology and pathophysiology. Classical techniques, in which polarographic oxygen electrode measures the extracellular oxygen concentration in a closed measuring vessel, require isolation and suspension of the cell. Because cell functions depend on the extracellular milieu including the extracellular matrix, isolation of cultured cells prior to the measurement may significantly affect the OCR. More recent techniques utilize optical methods in which oxygen-dependent quenching of fluorophores determines oxygen concentration in the medium at a few microns above the surface of the cultured cells. These techniques allow the OCR measurement in cultured cells adhered to the culture dish. However, this technique requires special equipment such as a fluorescence lifetime microplate reader or specialized integrated system, which are usually quite expensive. Here, we introduce a simple and inexpensive technique for measuring OCR in adherent cultured cells that utilizes conventional fluorescence microscopy and a glassware called a gap cover glass.

Keywords

Oxygen consumption Mitochondria Respiration Oxygen measurement 

Notes

Compliance with ethical standards

Conflict of interest

The authors declare no conflicts of interest.

Funding

Part of this work was supported by JSPS KAKENHI Grant Number 26430117.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. 1.
    Kramer PA, Ravi S, Chacko B, Johnson MS, Darley-Usmar VM (2014) A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: implications for their use as bioenergetic biomarkers. Redox Biol 2:206–210CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hagen T, Taylor CT, Lam F, Moncada S (2003) Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1α. Science 302:1975–1978CrossRefPubMedGoogle Scholar
  3. 3.
    Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC (2006) HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab 3:187–197CrossRefPubMedGoogle Scholar
  4. 4.
    Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7–25CrossRefPubMedGoogle Scholar
  5. 5.
    Agostini M, Romeo F, Inoue S, Niklison-Chirou MV, Elia AJ, Dinsdale D, Morone N, Knight RA, Mak TW, Melino G (2016) Metabolic reprogramming during neuronal differentiation. Cell Death Differ 23:1502–1514CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Semenza GL (2007) Oxygen-dependent regulation of mitochondrial respiration by hypoxia-inducible factor 1. Biochem J 405:1–9CrossRefPubMedGoogle Scholar
  7. 7.
    Horan MP, Pichaud N, Ballard JW (2012) Review: quantifying mitochondrial dysfunction in complex diseases of aging. J Gerontol A Biol Sci Med Sci 67:1022–1035CrossRefPubMedGoogle Scholar
  8. 8.
    Lestienne P (1999) Mitochondrial diseases. Models and methods. Springer, BerlinCrossRefGoogle Scholar
  9. 9.
    Schuh RA, Jackson KC, Schlappal AE, Spangenburg EE, Ward CW, Park JH, Dugger N, Shi GL, Fishman PS (2014) Mitochondrial oxygen consumption deficits in skeletal muscle isolated from an Alzheimer’s disease-relevant murine model. BMC Neurosci 15:24CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Schulze-Osthoff K, Bakker AC, Vanhaesebroeck B, Beyaert R, Jacob WA, Fiers W (1992) Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem 267:5317–5323PubMedGoogle Scholar
  11. 11.
    Souid AK, Tacka KA, Galvan KA, Penefsky HS (2003) Immediate effects of anticancer drugs on mitochondrial oxygen consumption. Biochem Pharmacol 66:977–987CrossRefPubMedGoogle Scholar
  12. 12.
    Sweet IR, Gilbert M, Scott S, Todorov I, Jensen R, Nair I, Al-Abdullah I, Rawson J, Kandeel F, Ferreri K (2008) Glucose-stimulated increment in oxygen consumption rate as a standardized test of human islet quality. Am J Transplant 8:183–192PubMedGoogle Scholar
  13. 13.
    Simonnet H, Vigneron A, Pouysségur J (2014) Conventional techniques to monitor mitochondrial oxygen consumption. Methods Enzymol 542:151–161CrossRefPubMedGoogle Scholar
  14. 14.
    Papas KK, Pisania A, Wu H, Weir GC, Colton CK (2007) A stirred microchamber for oxygen consumption rate measurements with pancreatic islets. Biotechnol Bioeng 98:1071–1082CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Frisch SM, Screaton RA (2001) Anoikis mechanisms. Curr Opin Cell Biol 13:555–562CrossRefPubMedGoogle Scholar
  16. 16.
    Danhier P, Copetti T, De Preter G, Leveque P, Feron O, Jordan BF, Sonveaux P, Gallez B (2013) Influence of cell detachment on the respiration rate of tumor and endothelial cells. PLoS One 8:e53324CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Papkovsky DB, Zhdanov AV (2016) Phosphorescence based O2 sensors—essential tools for monitoring cell and tissue oxygenation and its impact on metabolism. Free Radic Biol Med 101:202–210CrossRefPubMedGoogle Scholar
  18. 18.
    Yahara D, Yoshida T, Enokida Y, Takahashi E (2016) Directional migration of MDA-MB-231 cells under oxygen concentration gradients. Adv Exp Med Biol 923:129–134CrossRefPubMedGoogle Scholar
  19. 19.
    Dobrucki JW (2001) Interaction of oxygen-sensitive luminescent probes Ru(phen)32+ and Ru(bipy) 32+ with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements. J Photochem Photobiol B 65:136–144CrossRefPubMedGoogle Scholar
  20. 20.
    Kelbauskas L, Glenn H, Anderson C, Messner J, Lee KB, Song G, Houkal J, Su F, Zhang L, Tian Y, Wang H, Bussey K, Johnson RH, Meldrum DR (2017) A platform for high-throughput bioenergy production phenotype characterization in single cells. Sci Rep 7:45399. doi: 10.1038/srep45399 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Wagner BA, Venkataraman S, Buettner GR (2011) The rate of oxygen utilization by cells. Free Radic Biol Med 51:700–712CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Metzen E, Wolff M, Fandrey J, Jelkmann W (1995) Pericellular PO2 and O2 consumption in monolayer cell cultures. Respir Physiol 100:101–106CrossRefPubMedGoogle Scholar
  23. 23.
    Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nat Rev Cancer 11:325–337CrossRefPubMedGoogle Scholar
  24. 24.
    Semenza GL (2012) Hypoxia-inducible factors in physiology and medicine. Cell 148:399–408CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Elvidge GP, Glenny L, Appelhoff RJ, Ratcliffe PJ, Ragoussis J, Gleadle JM (2006) Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1α, HIF-2α, and other pathways. J Biol Chem 281:15215–15226CrossRefPubMedGoogle Scholar
  26. 26.
    Zhdanov AV, Okkelman IA, Collins FW, Melgar S, Papkovsky DB (2015) A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression. Biochim Biophys Acta 1847:1254–1266CrossRefPubMedGoogle Scholar

Copyright information

© The Physiological Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  1. 1.Advanced Technology Fusion, Graduate School of Science and EngineeringSaga UniversitySagaJapan

Personalised recommendations