Quantitative Metabolic Flux Analysis via 13C Labeling

To evaluate the quantitative metabolic flux in the cells, in vivo ¹³C labeling was performed as described previously [28 (link)] using NaH¹³CO₃ as a carbon source. Cultured cells were harvested and resuspended in MB6N containing 2% (w/v) sea salts and 25 mM NaH¹³CO₃ (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) to the same cell density as that of the culture. Time-course labeling was performed for 2, 4, and 10 min at illumination of 250 μmol m⁻² s⁻¹ white fluorescent lamps. Intracellular metabolites were analyzed using CE–MS as described above. ¹³C fraction, which is the ratio of ¹³C in total carbon, was calculated for each metabolite by detecting mass shifts from the ¹²C to ¹³C mass spectra.