Human fgf2

Rat ChondroSarcoma cells (RCS) were kindly provided by Dr Claudio Basilico from New York University School of Medicine. Cells were maintained in DMEM high glucose (Gibco) supplemented with 10% hiFBS (Gibco) and 1% Penicillin/Streptomycin/Glutamine (Gibco). For proliferation assay, 2500 cells were plated in black 96-well plate with optical bottom (Nunc). 24h after plating, medium was removed and replaced by assay medium (DMEM high glucose, 1% heat inactivated fetal bovine serum, 1% Penicillin/Streptomycin/Glutamine). Cells were either stimulated for 48h with increasing concentrations of human FGF2 (Peprotech) or 0,3ng/mL human FGF2 and increasing concentrations of Recifercept, both supplemented with 1μg/mL heparin sodium salt. Cell proliferation was assessed by measuring cell nuclei number with CyQuant Direct assay (Thermofisher Scientific). Fluorescence was measured with a Varioskan Lux plate reader.

Mouse embryos were harvested at E14.5, and the dorsolateral forebrain was dissected and enzymatically triturated to isolate a population of cells enriched in NPCs as previously described. NPCs isolated from a single brain were suspension-cultured in a T25 tissue culture flask in proliferation medium containing human EGF (10 ng ml^-1), human FGF2 (20 ng ml^-1) (Invitrogen, Carlsbad, CA), N2 supplement (1%) (GIBCO), penicillin (100 U ml^-1), streptomycin (100 mg ml^-1), and L-glutamine (2 mM) for 5 days and were allowed to proliferate to form neurospheres.

To prepare a neurosphere culture, the subventricular zone was dissected from neonatal mice and enzymatically dissociated with 0.25% trypsin and bovine DNase-I. Cells were passed through a 70 µm cell strainer followed by a 40 µm cell strainer and then plated with proliferation media consisting of neurosphere basal media (low-glucose DMEM, F12, HEPES, penicillin/streptomycin) supplemented with B27, mouse EGF, and human FGF2 (Invitrogen). To induce differentiation of neurospheres, growth factors were removed and N2 was added to the neurosphere basal media along with B27. Neurospheres were re-fed every 48 hr and used for up to 12 passages.
To infect neurospheres with LCMV, the cells were first plated in 24-well plates at a density of 125,000 cells/well on poly-d-lysine/laminin-coated coverslips (Invitrogen). The virus was diluted in neurosphere basal media and used to infect cells at 1 M.O.I. Infected cell cultures were incubated in a BSL-II primary tissue culture incubator at 37°C with 5% CO₂. To perform quantitative immunocytochemistry, cell cultures were fixed for 15 min at room temperature in 4% PFA and incubated in primary antibodies for 1 hr at room temperature. Secondary antibodies were then added for 1 hr at room temperature.