Glioblastoma Cell Culture and Transfection

Human glioblastoma cell lines U373 (cell line listed under catalogue number 89,081,403 has been re-named as U-251) and U87 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in DMEM high glucose medium (GE Healthcare, Chicago, IL, USA), supplemented with 10% (v/v) FBS, 2 mM l-glutamine, 100 IU/mL penicillin and 100 µg streptomycin. Glioblastoma stem cell line NCH421k was obtained from CLS (Cell Lines Service GmbH, Eppelheim, Germany) and expanded as spheroid suspensions in complete Neurobasal Medium (Invitrogen, Life Technologies, Carlsbad, CA, USA) containing 2 mM l-glutamine, 1 × penicillin/streptomycin, 1 × B-27 supplement (Invitrogen, Life Technologies, Carlsbad, CA, USA), 1 U/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/mL bFGF and EGF (both from Invitrogen, Life Technologies, Carlsbad, CA, USA). Cells were transfected with the plasmid vector pCMVDsRed-Express2 and pEGFP-N1 to stably express the red fluorescent protein DsRed and enhanced green fluorescent protein eGFP, as described previously [59 (link),63 (link)]. All cell lines were maintained at 37 °C with 5% CO₂ and 95% humidity. All cell cultures were tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).