Abstract
Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors—oligomycin to block ATP synthase, FCCP—to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A—to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.
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Acknowledgments
The authors thank Dr. Vladimir Gogvadze and Ganna Oliynyk for providing access to the equipment and help in setting up the initial protocol. This research was supported by Stiftelsen Samariten and Fredrik och Ingrid Thurings stiftelse.
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Smolina, N., Bruton, J., Kostareva, A., Sejersen, T. (2017). Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness. In: Gilbert, D., Friedrich, O. (eds) Cell Viability Assays. Methods in Molecular Biology, vol 1601. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6960-9_7
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DOI: https://doi.org/10.1007/978-1-4939-6960-9_7
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6959-3
Online ISBN: 978-1-4939-6960-9
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