OCR-Stats: Robust estimation and statistical testing of mitochondrial respiration activities using Seahorse XF Analyzer

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.

Vaping Perpetuates Cardiac Electrical Instability

“…Basal and ATP linked OCR were significantly depressed with apple jax and POG treatment versus air, but maximal OCR was significantly lower in air versus apple jax only (one way ANOVA, Bonferroni correction, p<0.05). As a whole, this experiment suggests that flavored e-liquids impair the cardiac myocyte's respiratory process 24 and an e-liquid with a higher toxicity depresses the respiratory processes more that an e-liquid with a lower toxicity.…”

“…We compared cellular respiration in HL-1 cells after 24h treatment with 25% e-vapor from an e-liquid with a higher toxicity (apple jax, APJ) or lower toxicity (POG) as determined in Figure 3. Oxygen consumption rate (OCR) was measured by the Agilent Seahorse 24 . Figure 4A is a graph of OCR versus time in 25% air, APJ or POG treated cells.…”

“…ATP-linked respiration was inhibited by addition of 1.5 μM oligomycin 24 (Oligo) then maximal respiration occurred after injection of 1.5 μM phenylhydrazone 24 (FCCP). The OCR remaining after addition of 0.5 μM rotenone + antimycin (A/R) is due to non-mitochondrial respiration 24 . Panel A curves indicated that apple jax depressed oxygen consumption more significant than air or POG (Kruskal-Wallis test with multiple comparisons, p<0.01).…”

“…Oxygen consumption rate measurement: The oxygen consumption rate (OCR) of HL-1 cardiomyocytes was measured using the XFp Cell Mito Stress Test by Agilent Seahorse 24 .…”

“…Cells were treated with 25% e-vapor extract for 24 h. Right before the measurement, the culture medium was replaced with the prewarmed assay medium (Agilent) containing (in mM) Glucose 10, Pyruvate 1, and L-Glutamate 200. Oligomycin, FCCP, and Rotenone/Antimycin are added to the cells in 20 minutes intervals, according to the essay's manufacturer's instruction in order to measure ATP linked, maximal, and non-mitochondrial oxygen consumption rates 24 . Soon after the experiment was completed, media was gently removed, and 40µl of RIPA lysis buffer was added, and cells were lysed using a 1 ml syringe.…”