Grey James F E, Townley Amelia R, Everitt Nicola M, Campbell-Ritchie Alistair, Wheatley Sally P
Faculty of Engineering, University of Nottingham, Nottingham, NG7 2UH, UK.
School of Life Sciences, University of Nottingham, Nottingham, NG7 2UH, UK.
Metabol Open. 2019 Nov 11;4:100020. doi: 10.1016/j.metop.2019.100020. eCollection 2019 Dec.
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.
细胞能量学分析对于理解包括糖尿病和癌症在内的代谢性疾病至关重要。监测细胞呼吸的两种最常用方法是海马XF系统和Glo™检测法，它们被视为“金标准”。这些商业方法间接测量能量学，并且需要大量资金投入。在此，我们描述了一种替代检测方法，它能够准确量化NADH周转率且成本低廉。该方法在存在纯化线粒体提取物的情况下，随着浓度升高测量刃天青还原为试卤灵的情况，直到NADH成为限速因子。这表明每个样品中NADH周转率的最大水平，从而推断代谢活性。在此，我们比较了具有不同代谢特征的MRC5、MCF7和MDA231细胞系。
