form one well of the 6-well plate were mechanical scraped into floating fragments, and the floating fragments were plated into non-coated T25 flask for floating culture with neural proliferation system II (N2B27 + 20 ng/mL bFGF + 20 ng/mL EGF, Thermo Fisher Scientific). Neurospheres usually formed on the second day of culture in neural proliferation system II. Accutase (StemPro® Accutase® Cell Dissociation Reagent, A1110501) was then used to detach neurosphere, and the single cells were plated in matrigel-coated dishes with neuron differentiation medium (N2, Thermo Fisher Scientific; B27, Thermo Fisher Scientific; 1 μM dibutyryl-cAMP, Sigma-Aldrich; 20 ng bdnf, PeproTech).
