Neurocult ns a proliferation supplement

We cultured three patient-derived GBM stem-like cell lines, kindly provided by S. Brandner (University College London, Division of Neuropathology, London, UK) in 6-well culture plates at 37 °C, 5% CO₂, and 100% humidity in NeuroCult NS-A basal medium supplemented with NeuroCult NSA proliferation supplement (1:10), 0.2% Heparin (1:1000), Hu Recom EGF (1:5000), Hu Recom bFGF (1:10,000; all from STEMCELL Technologies, Vancouver, Canada), laminin from Engelbreth-Holm Swarm murine sarcoma basement membrane (1:1000; Sigma Aldrich, St. Louis, MO, USA), and penicillin-streptomycin (1:100, 10,000 u/mL penicillin, 10,000 µg/mL streptomycin; Thermo Fisher Scientific, Waltham, MA, USA). We split the cells when they reached 80–90% confluency by collecting the media supernatant, washing the cells once with phosphate buffered saline (PBS; Biochrom, Berlin, Germany), collecting the PBS as well and then detaching the cells with Accutase (STEMMCELL Technologies, Vancouver, Canada). The detached cells and collected fluids were centrifuged for 10 min at 200× g. We discarded the supernatant, resuspended the cells in 1 mL medium and counted them using the Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA) before seeding 20,000 of them for staining experiments onto laminin-coated coverslips.

TS543 cells were plated at density 1 × 104 viable cells/cm² and grown as neurospheres with NeuroCult™ NS‐A basal medium supplemented with NeuroCult™ NS‐A proliferation supplement, 20 ng/ml EGF, 10 ng/ml bFGF, and 0.0002% heparin (Stem Cell Technologies). When diameters of neurospheres reached to approximately 100 μm, neurospheres were dissociated to single cells with mechanical force by pipetting 30–50 times.