A cost-effective, analytical method for measuring metabolic load of mitochondria.
A cost-effective, analytical method for measuring metabolic load of mitochondria.
复制标题
DOI：
10.1016/j.metop.2019.100020
复制
发表时间：
2019-12-01
期刊：
影响因子：
--
通讯作者：
Wheatley, Sally P
中科院分区：
文献类型：
--
作者：
Grey, James F E;Townley, Amelia R;Wheatley, Sally P
细胞能量学分析是了解包括糖尿病和癌症在内的代谢性疾病的核心。两种最常用的监测细胞呼吸的方法是SeaHorse-XF系统和Glo分析，这两种方法被认为是“金标准”。这些商业方法间接测量能量，需要大量的财政投资。在这里，我们描述了一种能够准确量化NADH周转的替代方法，而且这种方法是负担得起的。该方法测量在纯化的线粒体提取物存在下，Razurin还原为间苯二酚的浓度不断上升，直到NADH成为一个速率限制因素。这表明了每个样本中NADH周转的最大水平，从而推断出代谢活动。在这里，我们比较了具有不同代谢特征的MRC5、MCF7和MDA231 细胞系。
Analysis of cellular energetics is central to understanding metabolic diseases including diabetes and cancer. The two most commonly used methods to monitor cellular respiration are the Seahorse-XF system, and Glo assays, which are considered "gold standards". These commercial methods measure energetics indirectly and require considerable financial investment. Here we describe an alternative assay that enables accurate quantification of NADH turnover and that is affordable. This method measures resazurin reduction to resorufin at rising concentrations in the presence of purified mitochondrial extracts until NADH becomes a rate-limiting factor. This indicates the maximal level of NADH turnover in each sample and therefore infers metabolic activity. Here we compare MRC5, MCF7 and MDA231 cell lines which have differing metabolic profiles.