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Glucose labeling experiments in cell culture

YY Yan Yan
KK Karl N. Krecke
AB Aditi S. Bapat
TY Tingyuan Yang
ML Michael W. Lopresti
DM Douglas G. Mashek
AK Ameeta Kelekar
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Live, healthy cells were recovered by Ficoll density gradient centrifugation within 24 h prior to the experiment and 10 million cells per sample were pelleted and washed in a glucose-free medium. Cells were resuspended at 1E6 cells/ml in complete glucose-free medium supplemented with 10% dialyzed FBS, NEAA, and 4mM L-glutamine for 1 h and starvation and supplemented with 10 mM [1,2-13C] labeled glucose for 24 h. Cells were then pelleted and washed once with cold PBS and resuspended in 100–200 μl −20 °C methanol, snap-frozen, and stored at −80 °C. Liquid chromatography/mass spectrometry (LC/MS) and gas chromatography/mass spectrometry (GC/MS) were used for the identification and quantification of labeled metabolites of all samples. [1,2-13C] glucose was purchased from Cambridge Isotopes (Tewksbury, MA USA).

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