We applied a parallel labeling strategy using two isotopic tracers, [1,2-(13)C]glucose and [U-(13)C]glutamine, to determine metabolic fluxes in Chinese hamster ovary (CHO) cells. CHO cells were grown in parallel cultures over a period of six days with glucose and glutamine feeding. On days 2 and 5, isotopic tracers were introduced and (13)C-labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC-MS). Metabolites in glycolysis pathway reached isotopic steady state for [1,2-(13)C]glucose within 1 VSports手机版. 5h, and metabolites in the TCA cycle reached isotopic steady state for [U-(13)C]glutamine within 3h. Combined analysis of multiple data sets produced detailed flux maps at two key metabolic phases, exponential growth phase (day 2) and early stationary phase (day 5). Flux results revealed significant rewiring of intracellular metabolism in the transition from growth to non-growth, including changes in oxidative pentose phosphate pathway, anaplerosis, amino acid metabolism, and fatty acid biosynthesis. At the growth phase, de novo fatty acid biosynthesis correlated well with the lipid requirements for cell growth. However, surprisingly, at the non-growth phase the fatty acid biosynthesis flux remained high even though no new lipids were needed for cell growth. Additionally, we identified a discrepancy in the estimated TCA cycle flux obtained using traditional stoichiometric flux balancing and (13)C-metabolic flux analysis. Our results suggested that CHO cells produced additional metabolites from glucose that were not captured in previous metabolic models. Follow-up experiments with [U-(13)C]glucose confirmed that additional metabolites were accumulating in the medium that became M+3 and M+6 labeled. .