Seahorse XF Cell Mito Stress Test assay

Two 100 mm dishes were seeded with 2 × 10⁶ cells each and incubated with DHA or vehicle. After 24 h of incubation, cells were collected with trypsin–EDTA 0.25%, counted, and reseeded at 2 × 10⁴ density in Seahorse XF⁹⁶ V3 PS Cell Culture Microplates (#101085–004, Agilent Technologies Inc, USA) with the same medium to avoid changes in the DHA concentration. Then, the Seahorse XF Cell Mito Stress Test assay (#103015–100, Agilent Technologies Inc, USA) was performed according to the manufactures’ instructions. Briefly, Oligomycin (1 µM), FCCP (2 µM for PNT1A and 22Rv1; 0.5 µM for PC3) and Rotenone/Antimycin A (0.5 µM) were loaded at the cartridge ports. FCCP concentration and cell density per well were determined by prior titration experiments. Medium was removed from the microplate with the cells and replaced with warm and freshly prepared Seahorse XF Base Medium (#103681–100, Agilent Technologies Inc, USA), containing 1 mM pyruvate, 2 mM glutamine and 10 mM glucose (pH 7.4). Microplate coupled to the cartridge was placed at 37°C in non-CO₂ incubator for 45 min, followed by the determination of oxygen consumption (OCR) and Extracellular Acidification rates (ECAR) with Seahorse XFe96 Analyzer (Agilent Technologies Inc, USA). At the end of readings, cells in each well were lysed with RIPA buffer and total protein O.D. determined by BCA assay for normalization. Calculations were done by Wave software (v.2.6.3, Agilent) as follows: Basal Mitochondrial Respiration = (Late rate measurement before first injection) – (Non-mitochondrial respiration rate); Maximal Mitochondrial Respiration = (Maximum rate measurement after FCCP injection) – (Non-mitochondrial respiration rate); Proton Leak = (Minimum rate measurement after Oligomycin injection) – (Non-mitochondrial respiration); ATP-linked Production = (Late rate measurement before Oligomycin injection) – (Minimum rate measurement after Oligomycin injection); Spare Capacity = (Maximal Mitochondrial Respiration)—(Basal Mitochondrial Respiration); Non-mitochondrial respiration rate = Minimum rate measurement after Rotenone/ Antimycin A injection. Glycolytic Capacity was determined as ECAR rate upon Oligomycin injection. For statistical purpose, two independent experiments were performed with at least 10 replicates each (n = 20). Values were shown as mean of pmolO₂/minute/ protein for OCR or mpH/minute/protein for ECAR plus SEM.