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Cell culture and 13C-glucose labeling in vitro

RS Ramon C. Sun
TF Teresa W.-M. Fan
PD Pan Deng
RH Richard M. Higashi
AL Andrew N. Lane
AL Anh-Thu Le
TS Timothy L. Scott
QS Qiushi Sun
MW Marc O. Warmoes
YY Ye Yang
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An arsenite-transformed BEAS-2B (BAST) lung epithelial cell line36 was maintained in DMEM supplemented with 10 mM glucose, 2 mM Gln, 10% heat-inactivated FBS and 1% penicillin–streptomycin in 10 cm dishes. For the tracer experiment, BAST cells were allowed to reach  ~ 50% confluency, followed by the addition of DMEM base media supplemented with 10 mM 13C6-glucose, 2 mM Gln, 10% dialyzed fetal bovine serum, 50 U/mL penicillin, and 50 µg/ml streptomycin for 24 h in a CO2 incubator maintained at 37 °C. The enrichment in 13C6-glucose was > 99%. At the end of incubation, cells were washed with cold PBS three times followed by extraction with acetonitrile/water/chloroform (V/V 2:1.5:1) and separated into polar, lipid, and protein fractions35.

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