4.1. Culturing U2OS Cancer Cells

U2OS (human osteosarcoma) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; GibcoBRL, Grand Island, NY, USA), supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics (penicillin and streptomycin) at various initial concentrations of glucose and glutamine/L-alanine-glutamine (Table 1). Six of the compositions were made using Advanced DMEM that does not contain glucose, glutamine, phenol red or sodium pyruvate. Glc25Gln4 is commercially available (DMEM glutamaxTM). The cells were first cultured in DMEM glutamaxTM for two weeks until a sufficient number of cells was acquired. Then cells were split over seven experiments and cultured for three weeks to adopt. Cells cultured in conditions 3 and 4 started dying immediately.