Multinuclear MRS of Cell Cultures

U87 and NHA cells were grown on beads, BT142 neurospheres were encapsulated in agarose, and all MRS studies were performed using an MR-compatible cell perfusion system as described (29 (link)-31 (link)). All studies were performed on a 500MHz INOVA spectrometer (Agilent Technologies). For ¹³C-MRS studies glucose or glutamine in the medium were replaced with 5mM 1-¹³C-glucose or 2mM 3-¹³C-glutamine (Sigma-Aldrich) and spectra acquired using 60° flip angle (FA), 6s repetition time (TR) and 15min acquisition time. For hyperpolarized ¹³C-MRS, 2-¹³C-pyruvic acid (Sigma-Aldrich) was hyperpolarized using the Hypersense DNP polarizer (Oxford Instruments), injected into the perfusion system to a final concentration of 5mM pyruvate, and dynamic sets of ¹³C-MRS spectra acquired using 5° FA and 3s TR over 300s. All peak integrals were quantified using Mnova (Mestrelab Research). For thermally polarized ¹³C-MRS peaks, were normalized to cell number and initial ¹³C-substrate concentrations. For hyperpolarized ¹³C-MRS studies, total glutamate signal was normalized to total pyruvate signal and cell number.