Mox reagent

[U-13C6] glucose-containing medium was aspirated and cells were washed once with ice-cold saline, after which 500 μl of −20°C methanol was added to each well to quench metabolism. Next, 300 μl of ice cold ultrapure water containing 2 μg norvaline as an internal standard was added to a single well and cells were scraped into the methanol-water mixture and transferred to a microcentrifuge tube. This process was repeated for each well of the plate, after which 600 μl −20°C chloroform was added to each tube and all tubes were vortexed at 4°C for 10 min. The metabolite extracts were then centrifuged at 4°C at 17,200 x g for 10 min, and the resulting upper (polar metabolite) phases of the extracts were transferred to new tubes. These polar metabolite extracts were then dried in a centrifugal evaporator and stored at −80°C until derivatization for GC-MS analysis.
Metabolite extracts were derivatized by a two-step process. First, 15 μl of methoxyamine in pyridine (MOX Reagent, ThermoFisher) was added and the extracts were incubated at 40°C for 1.5 hr. Second, 20 μl N-(tert-butyldimethylsilyl)-N- methyl-trifluoroacetamide with 1% tert-butyldimethylchlorosilane (TBDMS) (Sigma) was added and samples were incubated at 60°C for 1 hr. The reaction mixtures were quickly vortexed and centrifuged, and supernatants were transferred to GC-MS vials for analysis.