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In fact, in non cross-over design, the placebo effect will not be mixed up with the effect of the test material

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Metabolic profiling Extraction and analysis of polar metabolites were performed as previously described 90,91 , with some modifications: 100 L of plasma were mixed with 1 mL of a pre-cooled (-20 C) homogenous methanol (MetOH):methyl-tert-butyl-ether (MTBE) 1:3 (v/v) mixture

Every batch verified 99% pure by RP-HPLC and identity-confirmed by LC-MS

On the subsequent days spheroids were washed in DMEM/F12 medium (Corning, USA) and the medium was changed, as follows: Basal medium: S1 MCDB131 (Corning, USA), 1% Glutamax (Thermo Fisher Scientific, Netherlands) + 1% Pen/Strep (Thermo Fisher Scientific, Netherlands), 2.44 mM glucose (Merck Millipore, USA), 29 mM sodium bicarbonate (Merck Millipore, USA), 2% BSA (Capricorn Scientific, USA), 2 l/100 mL ITS-X (Thermo Fisher Scientific, Netherlands)

Li XM, Huang CK, Zhang TF, Teper AA, Srivastava K, Schofield BH, et al