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 ageing. While associations between ageing and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from C. elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. Seahorse XF96 respirometers are the state-of-the-art platform to assess respiration in cells, and we adapted the technique for applications involving C. elegans. Here, we provide a detailed protocol to optimise and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes ~2 days to complete, excluding time spent culturing C. elegans, and includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparing and executing a run with the XF96 respirometer, and (iv) post-experimental data-analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the 8-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and high-throughput capacity due to 96-well format.

A novel ex vivo method for measuring whole brain metabolism in model systems

“…Previous studies have used the XFe96 to measure metabolism of C. elegans (Koopman et al, 2016). We measured larval stage 1 (L1), larval stage 4 (L4), and young adult day 1 (D1) worms using our micro-tissue restraints to demonstrate the versatility of this method, and to further validate our approach (Fig.…”

“…These L4 worms were then counted and added to the cell plates for analysis. Basal OCR rates were measured in M9 for 10 cycles according to the protocol described in Koopman et al , with each cycle consisting of two-minutes mixing, 30-seconds waiting, and two-minutes measuring (Koopman et al, 2016). After analysis, worms per well were counted again.…”

Enhancing mitochondrial proteostasis reduces amyloid-β proteotoxicity

“…Oxygen consumption was measured using the Seahorse XF96 equipment (Seahorse Bioscience) 34,50 . Respiration rates were normalized to the number of worms in each individual well and calculated as averaged values of 5 to 6 repeated measurements.…”

Self‐Destruction of Cancer Induced by Ag2S Amorphous Nanodots

“…Furthermore, the oxygen consumption rate (OCR), an indicator of mitochondrial respiration, was determined to evaluate the effects of different treatment on the mitochondrial function of cancer cells . From the results shown in Figure S9 of the Supporting Information, the basal respiration, maximal respiration, and spare capacity of cancer cells were calculated and the results were shown in Figure J–L.…”