A cost-effective, analytical method for measuring metabolic load of mitochondria

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.
Highlights • Gold standards for assessing metabolism are indirect and costly. • This paper provides a protocol to measure metabolic load of mitochondria that is direct and affordable. • By way of example, the metabolism of two breast cancer cell lines is compared.