Hypoxia Impacts on BCR-ABL Metabolome

CB or CB transduced BCR-ABL day 20 cells (+/- 24 hours of hypoxia) were used for NMR analyses. 20–30 million cells were quenched and the intracellular metabolites were extracted, evaporated using a SpeedVac concentrator and stored at 80°C until further analysis. For the NMR analysis dried samples were resuspended in 60 ml of 100mM sodium phosphate buffer containing 500 mM TMSP ((3-trimethylsilyl)propionic-(2,2,3,3-d4)-acid sodium salt) and 10% D₂O, pH 7.0. Samples were vortexed, sonicated and centrifuged briefly, before being transferred into a 1.7mm NMR tube using an automated Gilson sample handler. One-dimensional 1D ¹H-NMR spectra were acquired using a 600-MHz Bruker Avance III spectrometer (Bruker Biospin) with a TCI 1.7mm z-PFG cryogenic probe at 300 K. Each sample was automatically tuned, matched and then shimmed before acquisition of the spectrum. Spectra were processed using the MATLAB-based MetaboLab software [39 (link)]. The chemical shift was calibrated by referencing the TMSP signal to 0 ppm. Spectra were exported into Bruker format for metabolite identification and to determine concentrations using the Chenomx 7.0 software. The extracellular metabolites were measured directly in the used culture medium. All data presented here are in μMolar concentrations.