Glycolytic and Oxygen Consumption Rate Measurements (Seahorse).

Glycolytic rate of MCF7 and selected MCF7 cancer cells was measured using Seahorse XF96 extracellular flux analyzer and a glycolysis rate kit (Seahorse Biosciences). OCR and ECAR of cancer cells were determined by seeding them on XF96 microplates in their growth medium until they reached over 90% confluence. In these studies, seeding started with 20,000 cells (80% of well area). Measurements were determined 24 h later when the cells reached the 90% confluence. One hour before the Seahorse measurements, culture media were removed and cells were washed three times with PBS followed by addition of base medium (nonbuffered Dulbecco's Modified Eagle Medium supplemented with 25 mM glucose) or our nonbuffered only-glucose-containing solution. For glycolytic rate measurements, mitochondria inhibitors including rotenone (1 μM) and antimycin A (1 μM) were injected after basal measurements of ECAR and OCR of the cells under treatment to stop the mitochondrial acidification. 2-deoxy-glucose (100 mM) was added next to bring down glycolysis to basal levels. Finally, data were normalized for total protein content of each well using the Bradford protein assay (Thermofisher). Seahorse measurements were performed with four to six technical replicates, and these experiments were repeated four times.

A total of 2 mM Hepes, 2 mM MES, 5.3 mM KCl, 5.6 mM NaPhosphate, 11 mM glucose, 133 mM NaCl, 0.4 mM MgCl₂, and 0.42 mM CaCl₂ were titrated to the given pH with NaOH. For reduced Cl⁻ experiments, 133 mM NaCl was replaced with 133 NaGluconate, and MgCl₂ and CaCl₂ were raised to 0.74 and 1.46 mM, respectively, to account for gluconate-divalent binding. The amount of dilute HCl or NaOH added to medium to reduce pH to target level was determined empirically.

Respiratory capacity is a measure of the maximum rate of O₂ consumption and mitochondrial electron transport in a cell (46). Glycolytic capacity is the maximum rate of glucose conversion to pyruvate and/or lactate by a cell. Glucose breakdown to two lactates produces two protons, allowing for the capability of indirect measurement of glycolytic rate using the extracellular acidification (46). Compensatory glycolysis is the maximum possible rate of glycolysis in cells following inhibition of oxidative phosphorylation with rotenone/antimycin. The WE phenotype (“Warburgness”) can be expressed as the ratio of glycolysis (ECAR) to oxidative phosphorylation (expressed as the OCR) from the GST.