Culturing Glioblastoma Cell Lines for In Vivo Assays

Cell line U-87 MG was obtained from American Type Culture Collection (ATCC) and was cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin. Glioblastoma stem cell line NCH421k was purchased from Cell Lines Service (CLS) GmbH and cultured in Neurobasal Medium (Invitrogen, Waltham, MA, USA) supplemented with 2 mM L-glutamine, 1 × B-27 supplement (Invitrogen), 1 U/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/mL bFGF and EGF (both from Invitrogen, Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin. For in vivo assay, cells were transfected with plasmid vectors pCMVDsRed-Express2 and pEGFP-N1 to stably express red fluorescent protein DsRed and green fluorescent protein EGFP, as described previously [31 (link),32 (link)]. All cell lines were cultured at 37 °C, 5% CO₂ and 95% humidity and were tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).