Labeled DMEM medium was prepared from DMEM without glucose or glutamine (Sigma) by addition of 10 mM HEPES and the appropriate forms (labeled or unlabeled) of glucose and glutamine to a final concentration of 4.5 g l−1 glucose and 0.584 g l−1 glutamine (labeled glucose and labeled glutamine from Cambridge Isotope Laboratories), followed by sterile filtration. For kinetic flux profiling experiments, samples were switched to fresh unlabeled medium 1 h before the switch into 13C-labeled medium. This minimized metabolome perturbations at the time of the isotope switch resulting from removal of accumulated metabolic waste products. Metabolome quenching and extraction were conducted as previously described18 (link). Absolute metabolite quantitation involved extended labeling of cellular metabolites with uniformly labeled [13C]glucose and [13C]glutamine and extraction in the presence of known concentrations of unlabeled standards (for details, see refs. 23 (link),27 (link) and Supplementary Methods ).
Uptake of glucose and glutamine and excretion of all measured metabolites (excretion of pyruvate, lactate, alanine and glutamate were found to be significant) was determined from medium samples taken over an 8-h time period, centered at 48 h after infection. Glucose was measured by enzyme assay (E00715251, R-Biopharm). The other compounds were measured by LC-MS/MS, with inclusion of isotopic internal standards for glutamine, glutamate, pyruvate, lactate and alanine.
Estimation of the relative carbon flux between glycolysis and the PPP was carried out using [1,2-13C]glucose as described38 (link), with 4 h of incubation in labeled medium and detection of labeled forms of lactate by LC-MS/MS. Estimation of the relative carbon flux between pyruvate dehydrogenase and pyruvate carboxylase was carried out based on the passage of labeled carbon from [3-13C]glucose into malate, aspartate and citrate over 6 h (for details, seeSupplementary Methods ).
Uptake of glucose and glutamine and excretion of all measured metabolites (excretion of pyruvate, lactate, alanine and glutamate were found to be significant) was determined from medium samples taken over an 8-h time period, centered at 48 h after infection. Glucose was measured by enzyme assay (E00715251, R-Biopharm). The other compounds were measured by LC-MS/MS, with inclusion of isotopic internal standards for glutamine, glutamate, pyruvate, lactate and alanine.
Estimation of the relative carbon flux between glycolysis and the PPP was carried out using [1,2-13C]glucose as described38 (link), with 4 h of incubation in labeled medium and detection of labeled forms of lactate by LC-MS/MS. Estimation of the relative carbon flux between pyruvate dehydrogenase and pyruvate carboxylase was carried out based on the passage of labeled carbon from [3-13C]glucose into malate, aspartate and citrate over 6 h (for details, see