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Methods Mol Biol. 2017;1601:79-87. doi: 10.1007/978-1-4939-6960-9_7.

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

Author information

1
Karolinska Institutet, Stockholm, Sweden. natalia.smolina@ki.se.
2
Federal Almazov North-West Medical Research Centre, Akkuratova Str. 2, Saint Petersburg, 197341, Russia. natalia.smolina@ki.se.
3
Karolinska Institutet, Stockholm, Sweden.
4
ITMO University, Saint Petersburg, Russia.

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.

KEYWORDS:

Cell viability; Cellular respiration; Mitochondrial disease; Mitochondrial function; Myotubes; Satellite cells

PMID:
28470519
DOI:
10.1007/978-1-4939-6960-9_7
[Indexed for MEDLINE]

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