GBM Stem-like Cells Culture Protocol

Mouse 005 GBM stem-like cells (GFP positive) were provided by Dr. I Verma (Salk Institute) and have been described [21 (link), 24 (link)]. They were cultured as spheres in EF20 medium composed of Neurobasal medium (Thermo Fisher Gibco) supplemented with 3 mM l-Glutamine (Corning Mediatech), 1 × B27 supplement (Thermo Flasher Gibco), 0.5 × N2 supplement (Thermo Fisher Gibco), 2 μg/ml heparin (Sigma, St Louis, MO), 20 ng/ml recombinant human epidermal growth factor (R&D Systems, Minneapolis, MN), 20 ng/ml recombinant human fibroblast growth factor-2 (PeproTech, Rocky Hill, NJ), and 0.5 × penicillin G/streptomycin sulfate/amphotericin B complex (Corning Mediatech) at 37 °C and 5% CO₂. To passage cells, neurospheres were dissociated with the Neurocult chemical dissociation kit (Stem Cell Technologies). Mouse GBM GL261 cells were obtained from the National Cancer Institute and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were confirmed to be mycoplasma free (LookOut Mycoplasma kit, Sigma) and used at low passage number.