Glioblastoma Cell Culture Protocol

GBM cell lines, primary GBM cells, and normal human astrocytes were cultured as described previously (7 (link), 14 (link), 47 (link)). In brief, GBM cell lines A172, LN-18, SF-268, SF-295, T98MG, U251, and U87MG were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% EquaFETAL® bovine serum (Atlas Biologicals, Inc.) and 100 μg/ml streptomycin and 100 IU/ml penicillin (Gibco). Primary cells VTC-001, VTC-002, VTC-004, VTC-037, VTC-056, VTC-058, VTC-084, and VTC-103 were cultured in DMEM supplemented with 15% fetal bovine serum (Peak Serum, Inc.) and penicillin/streptomycin. Normal human astrocytes were cultured in MCDB-131 Medium (Sigma) containing 3% fetal bovine serum (Peak Serum, Inc.), 10 X G-5 Supplement (Gibco), and penicillin/streptomycin. Cell lines have been authenticated by the ATCC authentication service utilizing Short Tandem Repeat (STR) profiling. Primary GBM cells were kept at low passages (no more than 10). Antibodies of RAN and GAPDH were purchased from Santa Cruz Biotechnology, Inc. Importazole was purchased from Cayman Chemicals, Inc. Stock solution of importazole was prepared at 50 mM using dimethyl sulfoxide (DMSO). Working solution was further diluted using cell culture media.