Developers
Mandy Koopman, Helen Michels, Ellen
Description of the technology
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 studies. Mitochondrial respiration is recognized as an indicator of mitochondrial health. The Seahorse XF96 respirometer represents the state-
The technology proposes 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, XF96 application was compared with other existing techniques, including the
Practical application
On the basis of the comparisons with other respirometric methods, the XF respirometer approach offers important benefits for the measurement of mitochondrial respiration in C. elegans, especially when considering the ease of performing repeated measurements, comparing conditions and replicates, and investigating several aspects of mitochondrial function. This allows a higher throughput for
In vivo assessment of mitochondrial function in C. elegans also has, however, some clear limitations. First of all, oxygen consumption of an intact organism (e.g., basal respiration) does not strictly reflect mitochondrial respiration. For instance, cells possess a variety of oxygenases that also consume oxygen and thereby contribute to the OCR43. However, this disadvantage can be overcome by using of special compounds, e.g., residual respiration should be estimated in the presence of effective electron transport inhibitors in order to distinguish
This method can be used for
Laboratories
- Laboratory of Molecular Neurobiology of Ageing, European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen, Groningen (the Netherlands)
- Laboratory of Cardiac Energetics, National Heart, Lung and Blood Institute, Bethesda (USA)
- Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam (the Netherlands)
Links
http://www.nature.com/nprot/journal/v11/n10/full/nprot.2016.106.htmlPublications
- Koopman, M. et al «A
screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans." 11 Nature Protocols (2016): 1798–1816. - Andreux, P.A. et al. «A method to identify and validate mitochondrial modulators using mammalian cells and the worm C. elegans." 4 Sci. Rep. (2014): 05285.
- Houtkooper, R.H. et al. «Mitonuclear protein imbalance as a conserved longevity mechanism." 497 Nature (2013): 451–457.