Neurocult ns a basal medium

All fully established cancer lines were cultured at 37°C (5% (v/v CO₂) in vitro using RPMI supplemented with 10% (v/v) fetal calf serum and 10% (v/v) non‐essential amino acids. All primary human GBM cells were cultured at 37°C (5% (v/v) CO₂) in vitro using RPMI supplemented with 2% (v/v) fetal calf serum and 10% (v/v) non‐essential amino acids at 37°C (5% (v/v CO₂). GBM5/6/12/14 stem cells were cultured in StemCell Technologies NeuroCult NS‐A Basal Medium supplemented with 20 μg/ml bFGF, 20 μg/ml epidermal growth factor (EGF), and 2 mM heparin. CD133+ glioma cells from this population were isolated by fluorescence‐activated cell sorting analysis. Cells, for example, GBM12, grew as neurospheres and were characterized for multiple stem cell markers, including CD44, SOX2, CD133, CD15, CD36, Integrin B6, and MAP2. Neurosphere GBM cells had an approximate 10‐fold greater tumorigenicity in vivo than parental wild type (WT) GBM cells (data not shown). For short‐term cell killing assays and immunoblotting, cells were plated at a density of 3 × 10³ per cm² and 24 h after plating were treated with various drugs, as indicated. In vitro small molecule inhibitor treatments were from a 100 mM stock solution of each drug and the maximal concentration of vehicle (DMSO) in media was 0.02% (v/v). Cells were not cultured in growth factor free media during any study.