Neural stem cells are isolated from the subventricular zone of the adult brain of mice.
Single-cell suspensions are seeded at a density of 105 cells/ml in neurobasal medium, containing B27, N2, EGF (20 ng/ml), FGF (20 ng/ml), penicillin (100 U)/streptomycin (100 µg/ml). Cells are grown in uncoated T75 plastic flasks, as free-floating clusters (neurospheres). The cultures are passaged every 5–7 days. Cell isolation is performed by labeling with anti-LeX (SSEA-1) microbeads and purified by immunomagnetic selection. Positive cells are then selected for double positivity for LeX and CXCR4 by FACS.