66 Chapter 3 (2 mM). The manufacturer’s Seahorse XF glycolysis stress test protocol was used to assess glycolytic rate. After equilibration, sequential injections of glucose (20 mM), oligomycin (1µM), and 2-deoxy-D-glucose (50 mM) were performed. Glycolytic function is represented as glycolysis, glycolytic capacity, and glycolytic reserve. Glycolysis was calculated as the difference between the maximal rate before oligomycin addition and the last rate measurement before glucose addition. Glycolytic capacity was calculated as the difference between the maximal rate after oligomycin addition and the last rate measurement before glucose addition. The glycolytic reserve was calculated as the difference between glycolytic capacity and glycolysis. The measurements were normalized to the protein content in each well using a BCA assay. The same cell seeding density and treatments were used to measure mitochondrial respiration, measured as oxygen consumption rate (OCR). Cells were equilibrated for 1 hour in the absence of CO2 at 37 °C with XF base media, which was supplemented with glucose (20 mM), glutamine (2 mM), and pyruvate. The Seahorse XF cell mito stress test protocol was used to assess glycolytic rate. After equilibration, sequential injections of oligomycin (1µM), carbonyl cyanide-p- trifluoromethoxyphenylhydrazon (FCCP) (4µM), and rotenone/antimycin (1µM each) were performed. Mitochondrial respiration is represented as basal respiration, maximal respiration, spare respiratory capacity, spare capacity, ATP production, and proton leakage. Basal respiration is calculated as the difference between the last rate measurement before oligomycin addition and the minimum rate measurement after rotenone/ antimycin addition (which is defined as nonmitochondrial oxygen consumption). Maximal respiration is calculated as the difference between the maximum rate measurement after FCCP injection and the non-mitochondrial respiration. Spare respiratory capacity is calculated as the difference between maximal respiration and basal respiration. ATP production is calculated as the difference between the last rate measurement before oligomycin injection and the minimum rate measurement after oligomycin injection. The measurements were normalized to protein content in each well using a BCA assay following the manufacturer’s instructions (Pierce™ Microplate BCA Protein Assay Kit, Thermo Fisher Scientific). Tricarboxylic acid (TCA) cycle metabolites measurement by GC-MS Metabolite extraction and measurement methods were described by Bakker et al. (28). In short, cells were washed three times with ice-cold PBS and then quenched with methanol while being placed on ice. Ultrapure water was then added (with the addition of internal standards for quantification) and cells were removed from the flasks. Cell extracts were transferred into tubes containing chloroform. Methanol
RkJQdWJsaXNoZXIy MTk4NDMw