Abstract
Metabolic pathways and bioenergetics were described in great detail over half a century ago, and during the past decade there has been a resurgence in integrating these cellular processes with other biological properties of the cell, including growth control, protein kinase cascade signaling, cell cycle division, and autophagy. Since many disease conditions are associated with altered metabolism and production of energy, it is important to develop new approaches to measure these cellular parameters. This chapter summarizes a new and exciting approach based on the Seahorse XF24 Extracelluar Flux analyzer, which takes real time measurements of oxidative phosphorylation and glycolysis in living cells. These bioenergetic profiles are then compared with steady-state levels of cellular ATP as measured by a luciferase assay.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wu JJ, Quijano C, Chen E, Liu H, Cao L, Fergusson MM, Rovira II, Gutkind S, Daniels MP, Komatsu M et al (2009) Mitochondrial dysfunction and oxidative stress mediate the physiological impairment induced by the disruption of autophagy. Aging (Albany, NY) 1(4):425–437
Weinberg F, Hamanaka R, Wheaton WW, Weinberg S, Joseph J, Lopez M, Kalyanaraman B, Mutlu GM, Budinger GR, Chandel NS (2010) Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity. Proc Natl Acad Sci U S A 107(19): 8788–8793
Xiao D, Powolny AA, Moura MB, Kelley EE, Bommareddy A, Kim SH, Hahm ER, Normolle D, Van Houten B, Singh SV (2010) Phenethyl isothiocyanate inhibits oxidative phosphorylation to trigger reactive oxygen species-mediated death of human prostate cancer cells. J Biol Chem 285(34):26558–26569
Perez J, Hill BG, Benavides GA, Dranka BP, Darley-Usmar VM (2010) Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor. Biochem J 428(2):255–267
Hill BG, Dranka BP, Zou L, Chatham JC, Darley-Usmar VM (2009) Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal. Biochem J 424(1):99–107
Yao J, Irwin RW, Zhao L, Nilsen J, Hamilton RT, Brinton RD (2009) Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease. Proc Natl Acad Sci U S A 106(34):14670–14675
Roede JR, Jones DP (2010) Reactive species and mitochondrial dysfunction: mechanistic significance of 4-hydroxynonenal. Environ Mol Mutagen 51(5):380–390
Clark LC Jr (1956) Monitor and control of blood and tissue oxygen tensions. Trans Am Soc Artif Intern Organs 2:41
Bellance N, Benard G, Furt F, Begueret H, Smolkova K, Passerieux E, Delage JP, Baste JM, Moreau P, Rossignol R (2009) Bioenergetics of lung tumors: alteration of mitochondrial biogenesis and respiratory capacity. Int J Biochem Cell Biol 41(12):2566–2577
Tyystjarvi E, Ali-Yrkko K, Kettunen R, Aro EM (1992) Slow degradation of the d1 protein is related to the susceptibility of low-light-grown pumpkin plants to photoinhibition. Plant Physiol 100(3):1310–1317
Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J et al (2007) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 292(1):C125–C136
Nicholls DG, Johnson-Cadwell L, Vesce S, Jekabsons M, Yadava N (2007) Bioenergetics of mitochondria in cultured neurons and their role in glutamate excitotoxicity. J Neurosci Res 85(15):3206–3212
Yadava N, Nicholls DG (2007) Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone. J Neurosci 27(27):7310–7317
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Humana Press
About this protocol
Cite this protocol
de Moura, M.B., Van Houten, B. (2014). Bioenergetic Analysis of Intact Mammalian Cells Using the Seahorse XF24 Extracellular Flux Analyzer and a Luciferase ATP Assay. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_40
Download citation
DOI: https://doi.org/10.1007/978-1-62703-739-6_40
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-738-9
Online ISBN: 978-1-62703-739-6
eBook Packages: Springer Protocols