Neurobasal medium

Glioblastoma cell lines LN229 and U87 were purchased from the American Tissue Culture Collection (ATCC) (Manassas, VA). GBM cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA) supplemented with 10% FBS (HyClone, USA), 100 U/mL penicillin and 100 g/mL streptomycin (Gibco, USA). We obtained resected glioblastoma tumor biopsies from the UTHealth and Memorial Hermann, Texas Medical Center. The project was approved by both human subject research protection committees at UTHealth and University of Houston, and informed consent for participation in this study was obtained from each subject. All methods were performed in accordance with the relevant guidelines as described previously³⁴ . The patient-derived GBM tumor cells were cultured in Neurobasal Medium (StemCell Technologies, USA) supplemented with B27, N2, glutamax, heparin sulphate, hFGF and EGF. They were used up to passage 4. All cells were stored in a cell culture incubator at 5% CO₂, 37 °C.

Cortices of mice at embryonic day 14.5 (E14.5) for transfection and TrkB phosphorylation assays and E17.5 for immunostaining studies were collected in Hanks’ buffered salt solution (Sigma-Aldrich) and trypsinized in 1 mg/ml trypsin (Worthington) for 20 min at 37°C. The reaction was then stopped using 1 mg/ml trypsin inhibitor (Sigma-Aldrich) before the addition of 1 mg/ml DNase I (Thermo Fisher Scientific) and gentle dissociation with a 5 ml serological pipette. Cells were then pelleted by centrifugation at 1400 rpm for 5 min and resuspended in DMEM supplemented with 2% FBS, 1% GlutaMAX, and 1% Penstrep. Three hours after plating into wells coated with poly-d-lysine (Sigma-Aldrich), cells were maintained in Neurobasal medium supplemented with 1% GlutaMAX supplement, 1% Penstrep, and 2% SM1 supplement (Stem Cell Technologies). Neurons were cultured for up to 12 d with 50% media changes performed three times weekly. Subsequent transfections were performed on E14.5 neurons at 5DIV using 0.5 μg of indicated DNAs and 1 μl of Lipofectamine 2000 (see above). Depolarization of E17.5 neurons at DIV11 was achieved by supplementing media with 1 mm 4-aminopyridine (4-AP; Merck) for 24 h.