Human GBM neurosphere line, BT224-luc2 was donated by Dr Keith L Ligon of the Dana-Farber Cancer Institute, Boston, Massachusetts, USA; BT224-luc2 was derived from a patient at Brigham and Women’s Hospital undergoing surgery according to Institutional Review Board (IRB)-approved protocols, as previously described [56 (link),57 (link)]. Luciferase expression of human GBM neurosphere lines was performed using retrovirus encoding a fusion of luciferase and neomyocin phosphotransferase (pMMP-LucNeo), as previously described [58 (link)].
Human GBM neurospheres were cultured in Complete Human NeuroCultTM NS-A Proliferation Medium (Stemcell Technologies, Cambridge, UK) containing Human NeuroCultTM NS-A Proliferation Supplement (Stemcell Technologies, Cambridge, UK), rh-EGF (20 ng/mL; Miltenyi Biotec Ltd., Surrey, UK), rh-bFGF (10 ng/mL; Miltenyi Biotec Ltd., Surrey, UK), and 0.2% Heparin (2 μg/mL; Stemcell Technologies, Cambridge, UK). Human GBM neurospheres were grown in Corning Ultra-Low Attachment T25 cm2 or T75 cm2 Flasks (Sigma-Aldrich Ireland Ltd, Dublin, Ireland) and maintained at 37 °C in a 5% CO2 humidified incubator. Neurospheres were mechanically dissociated when they reached approximately 100–150 μm in diameter and passaged no more than twelve times.
Human GBM neurospheres were cultured in Complete Human NeuroCultTM NS-A Proliferation Medium (Stemcell Technologies, Cambridge, UK) containing Human NeuroCultTM NS-A Proliferation Supplement (Stemcell Technologies, Cambridge, UK), rh-EGF (20 ng/mL; Miltenyi Biotec Ltd., Surrey, UK), rh-bFGF (10 ng/mL; Miltenyi Biotec Ltd., Surrey, UK), and 0.2% Heparin (2 μg/mL; Stemcell Technologies, Cambridge, UK). Human GBM neurospheres were grown in Corning Ultra-Low Attachment T25 cm2 or T75 cm2 Flasks (Sigma-Aldrich Ireland Ltd, Dublin, Ireland) and maintained at 37 °C in a 5% CO2 humidified incubator. Neurospheres were mechanically dissociated when they reached approximately 100–150 μm in diameter and passaged no more than twelve times.