2.7. Oxygen consumption rate measurement (Mito Stress Test)

The Seahorse XF Extracellular Flux Analyser (Seahorse Bioscience Inc.) was used to measure the rate of mitochondrial oxidative phosphorylation using a fluorescent biosensor, which detects the oxygen consumption and glycolytic rates of cells seeded in microplates. On the day prior to the assay, cells were seeded in Seahorse XF-96 microplates at specific, previously determined densities (human dermal fibroblasts - 3 × 10⁵ per well and HeLa cells - 3 × 10⁴ per well, with 10–12 replicates per sample) and placed into an incubator overnight (5% CO₂, 37 °C). The provided sensor cartridge was hydrated with Seahorse XF Calibrant and placed at 37 °C in a non−CO₂ incubator overnight (or at least 5 h).

On the day of the assay, preparation of assay medium was performed by supplementing Seahorse XF base medium to achieve the following concentration: 10 mM glucose, 1 mM sodium pyruvate, 2 mM l-glutamine (pH of the assay medium was then adjusted to 7.4). The assay medium was placed into a water bath at 37 °C until ready for use. Injection compounds were prepared at the following stock concentrations: Oligomycin (100 μM), FCCP (100 μM) and rotenone/antimycin A (50 μM). Using a starting well volume of 175 μL, each compound was injected into the hydrated sensor plate (Oligomycin – Port A, FCCP – Port B, Rotenone/Antimycin A – Port C) at a volume of 25 μL with varying final well concentrations depending on cell type used. The sensor plate was placed into the analyser for calibration. The Seahorse XF Cell Culture Microplate was removed from the incubator and examined under the microscope to determine cell viability and desired confluence. Cells were supplemented with 175 μL of prepared assay medium and placed into a non−CO₂ incubator (37 °C) for 45 min to 1 h. After sensor plate calibration, the culture microplate was loaded into the analyser. Basal measurements of oxygen consumption and extracellular acidification rates were taken prior to and following injection of prepared compounds. Data retrieved was normalised to the protein concentration of each well determined by a Bradford assay (Bio-Rad).