2.3 The addition of DOX
2D monolayer culture: 2D cells were cultured at a seeding density of
3.5×105 cells/mL for 48 h. 3D MTS: HeLa cells were
cultured at a density of 2000 cells/well for 6 days. Then removed the
medium and incubated with 1μM DOX (dissolved in medium) for 48 h.
2.4 Preparation of uniformly13C-labeled cell extract
Pichia pastoris G/DSEL strains were cultivated with13C fully-labeled glucose as the sole carbon source,
and to increase the intracellular abundance of metabolites in the
central carbon metabolism, the 13C fully-labeled
glucose pulses were applied. The detailed operating procedures was
performed as described earlier [40]. The prepared cell extracts were
split and stored at -80°C pending for use. The extracts (100 μL)
mentioned above were added before intracellular metabolite extraction.
Based on the IDMS theory, this internal standard could track the loss of
metabolites during sample pretreatment and MS-based analytical process
[41].