Abstract
The accurate quantification of cellular and mitochondrial bioenergetic activity is of great interest in medicine and biology. Mitochondrial stress tests performed with Seahorse Bioscience XF Analyzers allow the estimation of different bioenergetic measures by monitoring the oxygen consumption rates (OCR) of living cells in multi-well plates. However, studies of the statistical best practices for determining aggregated OCR measurements and comparisons have been lacking. Therefore, to understand how OCR behaves across different biological samples, wells, and plates, we performed mitochondrial stress tests in 126 96-well plates involving 203 fibroblast cell lines. We show that the noise of OCR is multiplicative, that outlier data points can concern individual measurements or all measurements of a well, and that the inter-plate variation is greater than the intra-plate variation. Based on these insights, we developed a novel statistical method, OCR-Stats, that: i) robustly estimates OCR levels modeling multiplicative noise and automatically identifying outlier data points and outlier wells; and ii) performs statistical testing between samples, taking into account the different magnitudes of the between- and within-plate variations. This led to a significant reduction of the coefficient of variation across plates of basal respiration by 45% and of maximal respiration by 29%. Moreover, using positive and negative controls, we show that our statistical test outperforms the existing methods, which suffer from an excess of either false positives (within-plate methods), or false negatives (between-plate methods). Altogether, this study provides statistical good practices to support experimentalists in designing, analyzing, testing, and reporting the results of mitochondrial stress tests using this high throughput platform.
Highlights
Mitochondria are double-membrane-enclosed, ubiquitous, maternally inherited organelles present in most eukaryotic cells [1]
Using positive and negative controls from individuals known to have mitochondrial respiratory defects, we show that oxygen consumption rates (OCR)-Stats outperforms the currently used statistical tests, which suffer from an excess of either false positives or false negatives
We measured the OCR, the extracellular acidification rate (ECAR), and the cell number of 203 dermal fibroblast cultures derived from patients suspected to suffer from rare mitochondrial diseases and control cells from healthy donors
Summary
Mitochondria are double-membrane-enclosed, ubiquitous, maternally inherited organelles present in most eukaryotic cells [1]. They are known as the powerhouse of the cell [2,3] due to their pivotal function in the cellular energy supply where adenosine triphosphate (ATP) is generated by the mitochondrial respiratory chain in a process referred to as oxidative phosphorylation. One of the most informative tests of mitochondrial function is the quantification of cellular respiration, since it directly reflects electron transport chain impairment [9] and depends on many sequential reactions from glycolysis to oxidative phosphorylation [13]. The estimations of oxygen consumption rates (OCR) expressed in pmol/min enable drawing conclusions about the ability to synthesize ATP and about mitochondrial function, even more than the measurements of intermediates (such as ATP or nicotinamide adenine dinucleotide NADH) and potentials [14,15]
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