Poly l lysine and laminin

We obtained primary cortical neurons from E16 mice embryos. Briefly, the cortical tissue was dissected from embryonic brain tissue in ice-cold DMEM/F12 medium and digested in 0.05% trypsin for 15 min at 37°C. The cell density was adjusted to 5 × 10⁵/mL. Next, the cells were planted in poly-L-lysine and Laminin (Sigma–Aldrich, St. Louis, MO, USA) coated culturing ware in neurobasal medium with 2% B-27 supplement, 0.5% GlutaMAX-I^TM supplement, and 1% penicillin-streptomycin (Gibco, USA). The culture medium was half replaced every 3 days.

Murine GRP cells were received as a gift from the Department of Radiology and Radiological Science, Johns Hopkins School of Medicine (Baltimore, MD, USA). Cells were isolated as previously described by Srivastava, Bulte et al. [17 (link)] from spinal cords dissected from ^{Luc+/PLP/GFP+} mice between E12.5 and E14 stage. Tissue samples were plated on a Petri dish in DMEM/F12 medium (Thermo Fisher, Waltham, MA, USA), then incubated in pre-warmed TrypLE Express (Thermo Fisher, Waltham, MA, USA) with 10 mg/mL DNase-1 (A&A Biotechnology, Gdansk, Poland) for 10–12 min and incubated for a further 10 min. Next, 5 mL of GRP medium was added and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in 10 mL of GRP medium with 10 mg/mL of DNase, and incubated at 37 °C in a humidified incubator with 5% CO₂ for 10 min. The pellet was then mechanically agitated and centrifuged at 1000 rpm for 5 min; resuspended in 10 mL of GRP medium (DMEM/F12 medium (Thermo Fisher, Waltham, MA, USA); supplemented with N2 and B27 (Life Technologies, Carlsbad, CA, USA), 1% BSA (Abcam Cambridge, Great Britain), Penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA), and 20 ng per mL bFGF (Peprotech, Rocky Hill, NJ, USA)); and plated on coated with poly-L-lysine and laminin (Sigma, Saint Louis, MI, USA) 25 mL flasks in a humidified incubator at 37 °C with 5% CO₂.