Mitochondrial and Glycolytic Metabolism Assay

To evaluate the changes in mitochondrial and glycolytic function Seahorse XFp Cell MitoStress Test and Seahorse XFp Glycolysis Stress Test (Agilent Technologies, Santa Clara, CA) were performed according to the manufacturer’s protocol. In brief, MCF10A or MDA-MB-231 cells were plated at 40,000 cells/well in poly-D-lysine (50 µg/ml, Sigma-Aldrich) coated plates. The same number of cells were plated for each cell line. On the day of the assay the cell culture growth medium was replaced with either MitoStress or GlycoStress assay medium. MitoStress assay medium consisted of low-buffered pH 7.4 DMEM (Sigma-Aldrich) supplemented with glutamine (2 mM, ThermoFisher Scientific), glucose (10 mM, ThermoFisher Scientific) and pyruvate (1 mM, ThermoFisher Scientific). GlycoStress assay medium consisted of low-buffered pH 7.4 DMEM supplemented with glutamine (2 mM). The cell culture microplate was incubated in a non-CO2 incubator at 37 °C for 1 h prior to the assay. The seahorse compounds were prepared in assay media and injected into the injection ports.
For MitoStress assays, oligomycin (1 µM, Sigma-Aldrich), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 2 µM, Sigma-Aldrich) and rotenone/antimycin A (0.5 µM each, Sigma-Aldrich) were used. For glycolysis stress assays, glucose (10 mM), oligomycin (1 µM) and 2-deoxy glucose (50 mM, Sigma-Aldrich) were used.
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were reported as absolute rates (pmoles/min for OCR and mpH/min for ECAR). Data were exported from the Seahorse XFp Extracellular Flux Analyser into Seahorse XF Report Generator software. The nine replicates for each condition (3 technical replicates for each of the 3 biological replicates) were compiled in GraphPad Prism 7 Software.