Abstract
Mitochondrial function and dysfunction are at the core of aging and involved in many age-dependent diseases. Rate of oxygen consumption is a measure of mitochondrial function and energy production rate. The nematode Caenorhabditis elegans (C. elegans) offers an opportunity to study "living" mitochondria without the need for mitochondrial extraction, purification and associated artifacts. Oxygen consumption rate (OCR) is traditionally measured using single-chamber Clark electrodes with or without the addition of metabolic modulators. More recently, multi-well oxygen electrodes with automated injection system have been developed to enable rapid measurement of OCR under different conditions. Here, we describe a detailed protocol that we have adapted from existing protocols to measure coupled and uncoupled mitochondrial respiration (with and without metabolic modulators) in live respiring nematodes using a Seahorse XFe96 extracellular flux analyzer. We present details on our protocol, including preparation of nematode culture, use of metabolic modulators, execution of Seahorse XF assay as well as post-experimental data analysis. As a reference, we provide results of a series of experiments in which the metabolic activity of N2 wild-type nematodes was compared to N2 nematode treated with paraquat, a compound that generates reactive oxygen species (ROS), thus causing oxidative damage and mitochondrial dysfunction. These data illustrate the kind of insights that can be obtained even using a low number of nematodes (10 animals only per well).