2.5 Quenching and extraction procedures for intracellular metabolites
2D Hela cells were harvested after 48 h of incubation. The growth medium was removed and the flasks were washed by 5 mL PBS twice to remove extracellular metabolites. In all experiments, 2D cells were quenched on the flask surface and then scraped with extraction solvent. Cells were quenched and metabolites were extracted using 12 combinations of quenching and extraction methods as described below (Figure 1 ).
MTSs were harvested at 6th day and collected in 10 mL centrifuge tubes. The cell suspension was centrifuged at 1000×g for 5 min, the media was removed and the cell pellet was washed twice with 5mL PBS to remove extracellular metabolites. Then the MTSs were digested with Accutase™ cell digestive solution (40506ES60, Yesen, Shanghai, CN) at 37 ℃ for 30 min to form single-cell suspension and washed once with PBS. The single cell suspension was centrifuged for 10 min at 1000×g and the supernatant was removed.