3 13c glucose

For isotopic labeling experiments of fibroblasts, cells were cultured in DMEM medium supplemented with 25 mM [U-¹³C₆] or [3-¹³C]glucose (Cambridge Isotopes Inc), and 10% (v/v) dialyzed FBS for 48 hr prior to metabolite extraction. Metabolites abundance is shown as mole percent enrichment (MPE) time metabolite abundance per cell. MPE was calculated as the percent of all atoms within the metabolite pool that are labeled:
${\mathrm{\sum }}_{i=1}^n\frac{M_i·i}{n}$ where n is the number of carbon atoms in the metabolite and Mi is the relative abundance of the ith mass isotopomer. To measure the relative abundance of M1 labeled metabolites from [3-¹³C]glucose. PC3 cells were cultured in DMEM medium supplemented with 25 mM [U-¹³C₆]glucose (Cambridge Isotopes Inc) and 5% (v/v) dialyzed FBS as well as 0.5 mM glutamine, 0.5 mM asparagine, 0.1 mM free ammonium or without a nitrogen source for 4 days prior to metabolite extraction. For ¹⁵N labeling experiments PC3 cells were cultured in DMEM medium supplemented with 25 mM glucose and 5% (v/v) dialyzed FBS as well as 0.5 mM [γ-¹⁵N]glutamine (Cambridge Isotopes Inc), 0.5 mM [γ-¹⁵N]asparagine (Sigma) or 0.1 mM [¹⁵N]NH₄⁺ (Sigma) for 4 days prior to metabolite extraction.

Chemicals and M9 minimal medium were purchased from Sigma-Aldrich (St. Louis, MO). Isotopic tracers were purchased from Cambridge Isotope Laboratories (Tewksbury, MA): [1,6-¹³C]glucose (99.2 % ¹³C), [1,2-¹³C]glucose (99.7 %), [1-¹³C]glucose (99.5 %), [2-¹³C]glucose (99.5 %), [3-¹³C]glucose (99.5 %), [4,5,6-¹³C]glucose (99.5 %), and [1,2-¹³C]xylose (99.2 %). The isotopic enrichment of all tracers and the composition of tracer mixtures used in parallel labeling experiments were validated by GC-MS analysis as described in (Sandberg et al., 2016 (link)) and (Cordova and Antoniewicz, 2016 (link)). SFM4CHO medium (GE Healthcare Life Sciences SH3054901) and DMEM medium (Corning 17-207-CV, without glucose, glutamine, and sodium pyruvate) were purchased from Fisher Scientific (Pittsburgh, PA).

Culture materials were purchased from Cellgro (Mediatech, Manassas, VA). Chemicals were purchased from Sigma-Aldrich (St. Louis, MO). [1-¹³C]Glucose (99.5 atom% ¹³C), [2-¹³C]glucose (99.5%), [3-¹³C]glucose (99.5%), and [4,5,6-¹³C]glucose (99.8%) were purchased from Cambridge Isotope Laboratories (Andover, MA). Glucose stock solutions were prepared at 250 g/L in phosphate buffer saline (PBS). For experiments involving tracer mixtures, new glucose stock solutions were prepared by mixing the appropriate stock solutions at the desired ratio. The growth medium was Dulbecco’s modified Eagle medium (DMEM, Cat. No. 10-013-CV) supplemented with 10% fetal bovine serum (FBS, Cat. No. 35-011-CV) and 1% penicillin-streptomycin solution (PS, Cat No. 30-004-CI).