Metabolite Extraction and LC-MS Analysis

Cells were plated in triplicate plus an extra well for cell counting and cultured in standard medium for 18 h. Metabolite extraction buffer (MEB) was prepared composing of 50% methanol and 30% acetonitrile in water and kept at 4 °C. The counting well was then counted using the Invitrogen™ Countess™ automated cell counter based on manufacturer’s instructions to give the total number of cells per well. The media from the replicate wells was removed and the cells were washed once with 1xPBS. Metabolites were then extracted by adding 1 mL MEB per million cells and quickly scraped. The insoluble material was immediately pelleted in a cooled centrifuge (4 °C) at 13,000 rpm for 15 min and the supernatant was collected for subsequent LC–MS analysis. A HILIC column (4.6 × 150 mm, guard column 2.1 × 20 mm, Merck) was used for LC separation. The aqueous mobile phase solvent used was 0.1% formic acid in water (solvent A) and the organic mobile phase was 0.1% formic acid in acetonitrile (solvent B). The flow rate was set at 300 μl/min and the column oven set to 30 °C. The mobile phase gradient was described previously^{4 (link)}. Subsequent analysis was performed using the Xcalibur Quan Browser software (Thermo Fisher Scientific).