Glioblastoma Neurosphere Generation

The CT-2A-luc cells were generously donated by Dr. Martha R. Neagu. The cells were incubated at 37 °C with humidified air containing 5% CO₂. Monolayer CT-2A-luc cells were cultured in Dulbecco’s modified eagle medium with high glucose (DMEM; Thermo Fisher Scientific, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin-streptomycin. To generate neurospheres, CT-2A monolayer cells were enzymatically dissociated by accutase (Stem Cell Technology, San Diego, CA, USA) and seeded in 25 cm² culture dishes at the cell concentration of 1 × 10⁵ cells/mL in serum-free medium, composed of advanced DMEM/F12 medium (Life Technologies, Carlsbad, CA, USA) with l-glutamine (2 mM; Cellgro, Manassas, VA, USA), 1% N2 supplement (Life Technologies), 1% penicillin-streptomycin (Cellgro), recombinant EGF (20 ng/mL; R&D Systems, Minneapolis, MN, USA), and recombinant FGF2 (20 ng/mL; Peprotech, East Windsor, NJ, USA). After 10–11 days, the neurospheres were collected, enzymatically dissociated with accutase (Stem Cell Technology), and prepared for further studies [20 (link),21 (link)].