Guidelines for the measurement of oxygen consumption rate in Caenorhabditiselegans

Mitochondria are known as the powerhouse of the cell as through oxidative phosphorylation, they produce energy in the form of ATP. Nevertheless, mitochondria are also considered as the main producers of free radicals. Several mitochondrial parameters are needed to be examined to fully characterize mitochondria and the outcomes of their positive (i.e. energy production) or negative (i.e. production of free radicals/oxidative stress) function. Oxygen consumption rate (OCR) measurement is an excellent readout for mitochondrial respiratory capacity and it is the most frequently used assessment to examine mitochondrial function or as part of a broader bioenergetic profiling. Given the link between mitochondrial dysfunction, and increased oxidative stress and damage, and the fact that mitochondrial dysfunction is often reflected in OCR, its measurement is important for the complete characterization of the cellular redox status. Although much of this work is being done in cells or isolated mitochondria, there is an increasing need for the measurement of OCR in whole organismal models such as the nematode Caenorhabditis elegans. As a free-living organism with simple maintenance and conserved mitochondrial biology, C. elegans attracts interest as a model for ageing and age-related diseases, among others, in which bioenergetics but also various mitochondria-related redox aspects need to be evaluated. Therefore, the need for platforms suitable for OCR measurements in this model is evident. In this work, we have employed a newly developed system (Resipher) for the measurement of OCR in C. elegans and we outline basic protocols as well as the pharmacological interventions that can be used to assess the function of the respiratory chain. More specifically, we demonstrate the importance of the number of animals used in measurements that include mitochondrial complex inhibitors, how the presence of bacteria when used as a food source for the nematodes should be carefully considered and/or eliminated and how to avoid artefacts when measuring differently sized nematodes. The present work is not only intended to be used as a protocol for a specific measurement system but it can also be used as a guideline when setting up OCR experiments with any device, as it reveals parameters that may be overlooked and should be carefully considered.