Seahorse xfe96 extracellular flux analyser

Mitochondrial function in SHSY5Y cells was measured by using the Seahorse XFe96 Extracellular Flux Analyser (Agilent). For treatment with USP30Inh-1 8000 cells per well were seeded in the Seahorse 96 well plate and left to attach overnight. Next day, cells were treated with different USP30Inh-1 concentrations for 24 h. On the assay day, cell culture media was washed and replaced with fresh assay media (XF basic media; Agilent), supplemented with 10 mM glucose, 2 mM l-glutamine and 1 mM sodium pyruvate (pH was adjusted to 7.4). The plate was incubated at 37°C for equilibration for 1 h before loading to the analyser. Mitochondrial respiration was measured by using the Mito-Stress Test (Agilent) as per manufacturer's instructions. Oligomycin (1 µM), FCCP (1.2 µM), rotenone (1 µM) and antimycin A (1 µM) were sequentially added to cells to determine mitochondrial respiration parameters. For the normalisation step, 1 µg/ml Hoechst 33342 (Thermo Fisher) was added to the cells and incubated for 10 min before imaging on the PerkinElmer Opera Phenix.

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) represent the functions of mitochondria and glycolysis. Metabolic changes in the cells were analysed using a Seahorse XFe96 Extracellular Flux Analyser (Agilent Technologies, California, United States). 4×10³ cells per well were seeded into an XF 96 cell culture microplate (Agilent Technologies, California, United States). OCR and ECAR were assayed using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, California, United States, 103015–100) and Seahorse XF Glycolysis Stress Test Kit (Agilent Technologies, California, United States, 103020–100). Inhibitors and activators were used per well at the following concentrations via ports A–C: 1) ECAR including glucose (10 mM), oligomycin (1 μM) and 2-DG (50 mM); 2) OCR including oligomycin (1.5 μM), FCCP (0.5 μM) and Rot/AA (0.5 μM). Data were assessed using the Seahorse XF96 Wave software (Agilent Technologies, California, United States).

A total of 5 × 10³ cells per well were seeded into Agilent XFe96 Seahorse plates, transduced 24 h later with pAAV-ophNdi1 or pAAV-Ndi1, at an MOI of 3.4 × 10⁵. A mitochondrial stress test was performed 48 h later according to the manufacturer’s protocols (Seahorse XFe96 extracellular flux analyser, Agilent Technologies, CA, USA). Injection cycles were 5× for basal OCR, 5× following oligomycin (1 µM), 5× following FCCP (1 µM), 5× following rotenone (0.5 µM) and 5× following antimycin A (0.5 µM) injections [4 (link),49 (link)]. Six replicas per group were analysed. Basal and maximal OCRs, SRC and OCR rescue post-rotenone treatment (measurement 16–20/measurement 11–15 × 100) were determined. Primary fibroblasts were analysed as described above, but with 2.25 µM FCCP. The ATP Rate Assay was carried out as per the manufacturer’s protocol (Agilent Technologies, CA, USA) using 5 × 10³ cells per well. Seahorse analyses were normalised using the Pierce™ detergent compatible Bradford assay kit (Thermo Fischer Scientific, MA, USA), with absorbance determined at 595 nm (FLUOstar OPTIMA; BMG Labtech, Aylesbury, UK) and a standard test sample included on all Seahorse plates in a given experiment.