Metabolite Quantification in Cell Culture

After incubating cells in fresh media for 24 hr, the spent media were collected and analyzed. Known concentrations of isotope-labeled internal standards (glucose, lactate, glutamine, glutamate, palmitate, and aspartic acid; Cambridge Isotopes) were spiked into media samples before extraction. Extraction was performed with glass as previously reported (Yao et al., 2016b (link)). Samples were measured by LC/MS analysis, with the method described above. The absolute concentration of each compound was determined by calculating the ratio between the fully unlabeled peak from samples and the fully labeled peak from standards. The consumption rates (x) were normalized by cell growth over the experimental time period by using the following equation where N₀ represents the starting cell number, t represents incubation time, DT represents doubling time, and Y represents nutrient utilization. $Y={\int }_0^t{x\bullet N}_0\bullet 2^{t/DT}\bullet dt$