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Bioenergetic profile experiment using C2C12 myoblast cells - PubMed

  • ️Fri Jan 01 2010

Bioenergetic profile experiment using C2C12 myoblast cells

David G Nicholls et al. J Vis Exp. 2010.

Abstract

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity. Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO(2) into the extracellular environment. Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration--the Oxygen Consumption Rate--or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate--or ECAR. In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell. This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

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References

    1. Choi WS, Gerencser AA, Nicholls DG. Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure. J Neurochem. 2009;109:1179–1191. - PMC - PubMed
    1. Hill BG, Dranka BP, Zou L, Chatham JC, Darley-Usmar V. Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal. Biochem J. 2009;424:99–107. - PMC - PubMed
    1. Liu J, Cao L, Chen J, Song S, Lee IH, Quijano C, Liu H, Keyvanfar K, Chen H. Bmi1 regulates mitochondrial function and the DNA damage response pathway. Nature. 2009:459–7245. - PMC - PubMed
    1. Malmgren S, Nicholls DG, Taneera J, Bacos K, Koeck T, Tamaddon A, Wibom R, Groop L, Ling C, Mulder H, Sharoyko VV. Tight coupling between glucose and mitochondrial metabolism in clonal beta-cells is required for robust insulin secretion. J Biol Chem. 2009;284:32395–32404. - PMC - PubMed
    1. Dranka BP, Hill BG, Darley-Usmar VM. Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species. Free Radic Biol Med. 2010;48:905–914. - PMC - PubMed

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