LC-MS analysis of the cell metabolites

Cells with various treatments were cultured with Dulbecco's modified Eagle's medium (DMEM, Gibco, A14430-01) containing 4.5 g/L glucose, half of which was U-¹³C labeled (CIL, CLM-1396-1), for 1 hr. Then, 5 × 10⁶ cells were washed twice with cold PBS. After centrifugation, ice-cold extraction buffer (methanol: acetonitrile: H₂O = 2:2:1) was immediately added to the cell pellets at five volumes. Samples were treated with freeze and thaw cycles (freeze in liquid nitrogen for 1 min and thaw at room temperature) at least three times to lyse the cells sufficiently. Then, the same volume of chloroform was added to the samples and vortexed for 10 s. The mixtures were centrifuged at 12000 rpm for 15 min, and the supernatant was collected and dried. The powder containing the metabolites was dissolved in 80% methanol before running the LC-MS.