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Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer

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Mitochondria

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

Abstract

Energy homeostasis is critical for cellular function. Significant increases in energy demand or reduced energy supply, however, often result in cellular dysfunction and death. Since mitochondria are the primary cellular energy source, their impairment is often pathogenic. Accordingly, quantitative measurements of cellular and mitochondrial energy utilization and production are crucial for understanding disease development and progression. In the final step of cellular respiration, specifically, oxidative phosphorylation within the mitochondria, oxygen is consumed and drives ATP production. Herein, we provide the complete protocols for measuring oxygen consumption rates and their coupling to ATP production in intact and permeabilized cells, as well as in mitochondria isolated from tissue using the Seahorse XF Extracellular Flux Analyzer (Agilent Technologies).

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References

  1. Barbour JA, Turner N (2014) Mitochondrial stress signaling promotes cellular adaptations. Int J Cell Biol 2014:156020. https://doi.org/10.1155/2014/156020

    Article  CAS  Google Scholar 

  2. Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435(2):297–312. https://doi.org/10.1042/BJ20110162

    Article  CAS  Google Scholar 

  3. Li Z, Graham BH (2012) Measurement of mitochondrial oxygen consumption using a Clark electrode. Methods Mol Biol 837:63–72. https://doi.org/10.1007/978-1-61779-504-6_5

    Article  CAS  Google Scholar 

  4. Divakaruni AS, Paradyse A, Ferrick DA, Murphy AN, Jastroch M (2014) Analysis and interpretation of microplate-based oxygen consumption and pH data. Methods Enzymol 547:309–354. https://doi.org/10.1016/B978-0-12-801415-8.00016-3

    Article  CAS  Google Scholar 

  5. Rogers GW, Brand MD, Petrosyan S, Ashok D, Elorza AA, Ferrick DA, Murphy AN (2011) High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One 6(7):e21746. https://doi.org/10.1371/journal.pone.0021746

    Article  CAS  Google Scholar 

  6. Gerencser AA, Neilson A, Choi SW, Edman U, Yadava N, Oh RJ, Ferrick DA, Nicholls DG, Brand MD (2009) Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal Chem 81(16):6868–6878. https://doi.org/10.1021/ac900881z

    Article  CAS  Google Scholar 

  7. Divakaruni AS, Rogers GW, Murphy AN (2014) Measuring mitochondrial function in permeabilized cells using the seahorse XF analyzer or a Clark-type oxygen electrode. Curr Protoc Toxicol 60:25.2.1–25.216. https://doi.org/10.1002/0471140856.tx2502s60

    Article  Google Scholar 

  8. Salabei JK, Gibb AA, Hill BG (2014) Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat Protoc 9(2):421–438. https://doi.org/10.1038/nprot.2014.018

    Article  CAS  Google Scholar 

  9. Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17:65–134. https://doi.org/10.1002/9780470122624.ch2

    Article  CAS  Google Scholar 

  10. Pfleger J, He M, Abdellatif M (2015) Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival. Cell Death Dis 6:e1835. https://doi.org/10.1038/cddis.2015.202

    Article  CAS  Google Scholar 

  11. Jeon YH, He M, Austin J, Shin H, Pfleger J, Abdellatif M (2021) Adiponectin enhances the bioenergetics of cardiac myocytes via an AMPK- and succinate dehydrogenase-dependent mechanism. Cell Signal 78:109866. https://doi.org/10.1016/j.cellsig.2020.109866

    Article  CAS  Google Scholar 

  12. Schnaitman C, Greenawalt JW (1968) Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol 38(1):158–175. https://doi.org/10.1083/jcb.38.1.158

    Article  CAS  Google Scholar 

  13. Pfleger J, Gross P, Johnson J, Carter RL, Gao E, Tilley DG, Houser SR, Koch WJ (2018) G protein-coupled receptor kinase 2 contributes to impaired fatty acid metabolism in the failing heart. J Mol Cell Cardiol 123:108–117. https://doi.org/10.1016/j.yjmcc.2018.08.025

    Article  CAS  Google Scholar 

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Acknowledgments

I thank Dr. Maha Abdellatif (Rutgers University—Newark, NJ) for the advice, discussions and support.

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Correspondence to Jessica Pfleger .

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Pfleger, J. (2022). Measurements of Mitochondrial Respiration in Intact Cells, Permeabilized Cells, and Isolated Tissue Mitochondria Using the Seahorse XF Analyzer. 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_12

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  • DOI: https://doi.org/10.1007/978-1-0716-2309-1_12

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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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