We used previously established primary brain tumor initiating cell lines (BTIC)-10 and -12 derived from patients with IDH wild type glioblastoma as described15 (link),41 . The Department of Neuropathology, University of Regensburg (MJR), verified the patients’ diagnoses and WHO grade.
Tumor cells were maintained in RHB-A (Y40001, Takara), supplemented with 20 ng/ml of EGF (130097751), bFGF (130093842) (both Miltenyi Biotech), and 50 U (v/v) Penicillin/0.05% (v/v) Streptomycin (P4333) (Sigma-Aldrich) at 37 °C, 5% CO2, 95% humidity in a standard tissue culture incubator. Progenitor features of BTICs were verified by clonogenicity assays, flow cytometry (CD133, CD15, CD44, A2B5), immunocytochemistry (Nestin, Sox2, GFAP), and tumor take in an immunocompromised mouse model (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BTICs were lentivirally transduced to achieve stable expression of the nuclear fluorescent protein H2B Dendra2, as described61 (link).
The ethics board of the University of Regensburg, Germany, approved the use of human material for this study (No° 11-103-0182) and all patients gave written informed consent. All methods were performed in accordance with the relevant guidelines and regulations.
Tumor cells were maintained in RHB-A (Y40001, Takara), supplemented with 20 ng/ml of EGF (130097751), bFGF (130093842) (both Miltenyi Biotech), and 50 U (v/v) Penicillin/0.05% (v/v) Streptomycin (P4333) (Sigma-Aldrich) at 37 °C, 5% CO2, 95% humidity in a standard tissue culture incubator. Progenitor features of BTICs were verified by clonogenicity assays, flow cytometry (CD133, CD15, CD44, A2B5), immunocytochemistry (Nestin, Sox2, GFAP), and tumor take in an immunocompromised mouse model (female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BTICs were lentivirally transduced to achieve stable expression of the nuclear fluorescent protein H2B Dendra2, as described61 (link).
The ethics board of the University of Regensburg, Germany, approved the use of human material for this study (No° 11-103-0182) and all patients gave written informed consent. All methods were performed in accordance with the relevant guidelines and regulations.
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