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
Barbour JA, Turner N (2014) Mitochondrial stress signaling promotes cellular adaptations. Int J Cell Biol 2014:156020. https://doi.org/10.1155/2014/156020
Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435(2):297–312. https://doi.org/10.1042/BJ20110162
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
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
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
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
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
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
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
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
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
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
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
Acknowledgments
I thank Dr. Maha Abdellatif (Rutgers University—Newark, NJ) for the advice, discussions and support.
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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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