Bioenergetic profile experiment using C2C12 myoblast cells.
Bioenergetic profile experiment using C2C12 myoblast cells.
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DOI：
10.3791/2511
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发表时间：
2010-12-06
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影响因子：
--
通讯作者：
Ferrick, David A
中科院分区：
文献类型：
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作者：
Nicholls, David G;Darley-Usmar, Victor M;Ferrick, David A
测量细胞代谢以及了解线粒体功能障碍的能力，使世界各地的科学家能够推进他们在理解线粒体功能在肥胖、糖尿病、衰老、癌症、心血管功能以及安全毒性方面所起作用的研究。细胞代谢是底物摄取（如氧气、葡萄糖、脂肪酸和谷氨酰胺）以及随后通过一系列酶控氧化和还原反应进行能量转换的过程。这些细胞内的生化反应导致三磷酸腺苷（ATP）的产生、热量的释放以及化学副产物（如乳酸和二氧化碳）释放到细胞外环境中。通过测量细胞消耗氧气的速率（线粒体呼吸的一个指标——耗氧率，即OCR），可以深入了解细胞的生理状态以及这些细胞状态的改变。细胞也通过糖酵解产生ATP，即葡萄糖转化为乳酸，这一过程不依赖氧气。在培养孔中，乳酸是质子的主要来源。通过测量细胞周围细胞外介质中释放的质子间接产生的乳酸（这会导致介质酸化），可得到细胞外酸化率，即ECAR。在本实验中，将C2C12成肌细胞以给定的密度接种在海马细胞培养板中。测量基础耗氧率（OCR）和细胞外酸化率（ECAR）以确定基线速率。然后通过依次添加三种不同的化合物对细胞进行代谢扰动，从而改变细胞的生物能量特征。该实验方法源自一个评估线粒体的经典实验，并作为一个框架，在此基础上构建更复杂的实验，旨在了解线粒体的生理和病理生理功能，并预测细胞应对压力和/或损伤的能力。
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.