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A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans

Abstract

Mitochondrial dysfunction is at the core of many diseases ranging from inherited metabolic diseases to common conditions that are associated with aging. Although associations between aging and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from Caenorhabditis elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. The Seahorse XF96 respirometer represents the state-of-the-art platform for assessing respiration in cells, and we adapted the technique for applications involving C. elegans. Here we provide a detailed protocol to optimize and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes 2 d to complete, excluding the time spent culturing C. elegans, and it includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparation and execution of a run with the XF96 respirometer and (iv) postexperimental data analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the eight-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and the high throughput capacity due to the 96-well format.

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Figure 1: A schematic overview of glycolysis and oxidative phosphorylation.
Figure 2: The measurement units of XF respirometers.
Figure 3: Distinguishing non-mitochondrial respiration from mitochondrial respiration.
Figure 4: Temperature does not influence the OCR of C. elegans.
Figure 5: A schematic overview of an XF respirometer experiment.
Figure 6: Respiration rates change during development and with age in C. elegans.
Figure 7: Typical example of OCR profiles in C. elegans respirometry.
Figure 8: Detailed tracing of oxygen levels is required to identify experimental issues.
Figure 9: The OCR correlates with the number of worms.
Figure 10: The effects of specific compounds on the OCR in C. elegans.
Figure 11: Genetic manipulation of the electron transport chain lowers the OCR in C. elegans.
Figure 12: XFp respirometers provide an alternative to XF96 respirometers.

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Acknowledgements

We thank T. Ackermann for advice and C.F. Calkhoven for allowing us to make use of the Seahorse XF96 machine. Work in the Nollen group is financially supported by an European Research Council (ERC) starting grant (no. 281622). Work in the Houtkooper group is financially supported by an ERC starting grant (no. 638290) and a VIDI grant from ZonMw (no. 91715305). The Seahorse XF96 at the Academic Medical Center, Amsterdam, the Netherlands, was supported by a grant from NWO-Middelgroot (no. 91112009).

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Contributions

M.K. performed most of the experiments, optimized the final experimental pipeline and wrote the manuscript together with R.H.H., and with contributions from all authors. H.M. and R.K performed experiments using the XFp/XF96 respirometers. L.M., R.H.H., B.M.D. and J.A. pioneered the use of XF respirometers with C. elegans and developed the initial protocols. M.K., H.M., E.A.A.N., B.M.D. and R.H.H. have been extensively involved in discussions and interpretations of results.

Corresponding authors

Correspondence to Mandy Koopman or Riekelt H Houtkooper.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Titration of antimycin A and rotenone

(a) Titration of Antimycin A, n = 8-12 wells, and (b) Antimycin A & Rotenone, 8-16 wells, does not decrease the OCR. One-way ANOVA (ns). Bars are mean ± SEM.

Supplementary Figure 2 Respiration rate of E.coli.

Respiration rates of 15 worms compared to E. coli measured with a XFp respirometer. The respiration of E. coli is clearly lower than the respiration of worms. Bars are mean ± SEM.

Supplementary Figure 3 XF96 plate layout

This figure shows a representative plate layout that can be filled in for pre- and post-experimental preparation and analysis.

Supplementary Figure 4 Respiration correlates with the number of worms

Linear regression of the OCR as dependent variable of the number of worms in (a) L4s, (b) Adult day 3, (c) Adult day 5 and (d) Adult day 8. Grey area shows the confidence interval (95%). The dotted line in a-d shows the linear regression, n = 44-48 in a-d.

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Supplementary Figures 1–4 (PDF 542 kb)

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Koopman, M., Michels, H., Dancy, B. et al. A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans. Nat Protoc 11, 1798–1816 (2016). https://doi.org/10.1038/nprot.2016.106

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