Culturing CT-2A Glioma Stem Cells

CT-2A cells were provided by Prof. Thomas Seyfried (Boston College, Boston, MA, USA) (Seyfried et al., 1992 (link)). Cells were incubated at 37°C in humidified air with 5% CO₂. ML/CT-2A cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific), supplemented with 10% heat-inactivated fetal calf serum (FCS; Thermo Fisher Scientific) (Seyfried et al., 1992 (link)). To generate NS/CT-2A cells, we adapted a previously published protocol (Binello et al., 2012 (link)). Briefly, confluent ML were enzymatically dissociated (Stempro Accutase, Thermo Fisher Scientific) and cells were plated at 1×10⁵ cells/ml in DMEM/Nutrient Mixture F-12 (DMEM/F-12; Thermo Fisher Scientific) supplemented with 20 ng/ml epidermal growth factor (EGF; Thermo Fisher Scientific), 20 ng/ml fibroblast growth factor (FGF; Thermo Fisher Scientific) and 2% B27 supplement (Thermo Fisher Scientific). Six days after plating, NS were collected, enzymatically dissociated and re-plated at the same initial concentration. Two and 8 days after the start of the NS culture, the medium was supplemented with 20 ng/ml EGF and FGF. Eleven days after the start of the culture, NS were collected, enzymatically dissociated and prepared for further applications.