Xf96 extracellular flux analyser

Oxygen consumption rate (OCR) in fibroblasts was measured with a XF96 Extracellular Flux Analyser (Seahorse Bioscience, Agilent Technologies, Cheshire, UK), as described previously (37 (link)). Each cell line was seeded in 12 wells of a XF96-well cell culture microplate (Seahorse Bioscience) at a density of 30×10³ cells/well in 80 µl of DMEM and incubated for 24 h at 37°C in 5% CO₂ atmosphere. After replacing the growth medium with 180uL of bicarbonate-free DMEM (pre-warmed at 37°C), cells were pre-incubated for 30 min before starting the assay procedure. Oxygen consumption rate (OCR), leaking respiration (LR), maximal capacity respiration (MCR) and not electron transport chain respiration (NMR) were determined by adding 1 μM oligomycin (LR), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (MCR: 2 injections of 0.5 μM and 1 μM, respectively) and 1μM Rotenone/antimycin (NMR), respectively (all Sigma-Aldrich, Dorset UK). The data were corrected by the NMR and expressed as pmol of oxygen/min/mg of protein. The quantity of protein was measured by Bradford assay.

Controls and patients’ fibroblasts were seeded at 7000 cells/well in 100 μl of medium (RPMI/Amniomax) in Seahorse XF96 Cell Culture Microplates coated with 1/100 dilution Corning Matrigel hESC-qualified matrix (Dominique Dutscher) and using 8 replicates. Cells were incubated for 48 hours at 37°C in a 5% CO₂ atmosphere.
Cellular oxygen consumption (oxygen consumption rate [OCR]) was assayed using the Seahorse XF96 Extracellular Flux Analyser with sequential addition of oligomycin (0.5 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1 μM), rotenone (1 μM), and antimycin (0.05 μM) to measure basal and maximal respiration. OCR was normalized to protein content. For mitochondrial complexes I and II, respiratory rates were measured as previously described (35 (link)).

The real-time mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the XF96 Extracellular Flux Analyser (Seahorse Bioscience) with the Cell Mito Stress Kit (Seahorse), as per the manufacturer’s instructions. MDA-MB-468 cells in osteogenic differentiation for 5 days and undifferentiated control cells were used for metabolic analysis. About (8–10) × 10³ cells per well were seeded in XFe96 cell culture plates and incubated for 24 h in F medium at 37 °C in a humidified atmosphere with 5% CO₂. Before the assay, cells were rinsed twice and placed in a prewarmed XF assay medium (pH 7.4) at 37 °C in a non-CO₂ incubator for 1 h. Subsequently, for measurement of ECAR, 10 mM glucose, 1 µM oligomycin and 50 mM 2-DG in XF assay medium were loaded into the injection ports in the XFe96 sensor cartridge to determine the ECARs. For measurement of mitochondrial oxygen consumption, the respiratory rate was measured at 37 °C by using the following perturbation drugs in succession: 1 µM oligomycin, 2 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone and 0.5 µM rotenone/antimycin A. The basal OCR was measured before drug exposure. The experiment was performed with at least three replicates per condition.

The mitochondrial integrity was assessed by measuring the mitochondrial membrane potential (Δψ) using the fluorescent dye JC‐1 (BD, USA) according to the manufacturer's protocol. The samples were analysed using flow cytometry.
Real‐time monitoring of the cellular oxygen consumption rate (OCR) was performed using an XF96 extracellular flux analyser (Seahorse Bioscience) following the manufacturer's instructions. During the measurement, the following inhibitors of the respiratory chain components were serially added to the culture medium: the ATP synthase inhibitor oligomycin (1 µM); the respiratory uncoupler FCCP (0.5 µM); and the complex I and III inhibitors rotenone (0.5 µM) and antimycin A (0.5 µM).