A novel C. elegans respirometry assay using low-cost optical oxygen sensors

Abstract

Measurement of the oxygen consumption rate (OCR), or respirometry, is a powerful and comprehensive method for assessing mitochondrial function both in vitro and in vivo. Respirometry at the whole-organism level has been repeatedly performed in the model organism Caenorhabditis elegans, typically using high throughput microplate-based systems over traditional Clark-type respirometers. However, these systems are highly specialised, costly to purchase and operate, and inaccessible to many researchers. Here, we develop a respirometry assay using low-cost commercially available optical oxygen sensors (PreSens OxoPlates(R)) and fluorescence plate readers (the BMG FLUOstar), as an alternative to more costly standard respirometry systems. This assay uses standard BMG FLUOstar protocols and a set of custom scripts to perform repeated measurements of the C. elegans OCR, with the optional use of respiratory inhibitors or other interventions. We validate this assay by demonstrating the linearity of basal OCRs in samples with highly variable numbers of animals, and by examining the impact of respiratory inhibitors with previously demonstrated efficacy in C. elegans: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (a mitochondrial uncoupler) and sodium azide (a Complex IV inhibitor). Using this assay, we demonstrate that the sequential use of FCCP and sodium azide leads to an increase in the sodium azide-treated (non-mitochondrial) OCR, indicating that the sequential use of respiratory inhibitors, as standard in intact cell respirometry, may produce erroneous estimates of non-mitochondrial respiration in C. elegans and thus should be avoided.