Neurosphere culture and in vitro assay

 Anesthetize pregnant mice with CO₂ gas and collect embryos from the uterus. If knockout mice are used for the analyses, cut tails of embryos for genotyping.

 After cutting head skin and opening a skull, remove brains and place them into cold L15 medium.

 Under a dissecting microscope, open the brains along the midline of cerebral cortex to cut out ganglionic eminences (GE in Fig. 1) from the left and right semi-sphere of the forebrain. Meninges and visible blood vessels should be carefully removed from the section.

 Place GE into 0.5 mL DME:F-12/B27 medium and dissociate GE into single cells mechanically by pipetting with a polished Pasteur pipette.

 Add 4 mL DME:F-12/B27 medium and stand the tubes for 5 min to precipitate non-dissociated cells.

 Transfer cells except precipitate into a new tube and centrifuge at 300 × g for 5 min. Resuspend the precipitated cells with DME:F-12/B27 medium supplemented with 20 ng/mL FGF2 and 20 ng/mL EGF as mitogens and antibiotics in a tissue culture flask.

 To keep cells growing, add FGF2 and EGF into the culture medium every other day.

 At the 6th or 7th day in vitro, the floating neurosphere cells are precipitated and dissociated by pipetting as described above. This step is repeated until neurosphere cells are used for in vitro assays.

 Coat 12-well plates with 0.01% polyethylenimine for 2 h. Wash the wells with DDW twice and once with PBS.

 Then, coat the plates with 5 μg/mL mouse laminin for 2 h at 37°C. Wash the wells with PBS 4 times.

 Add 500 μL Neurobasal/B27 medium into each well and keep the plates in CO₂ incubator.

 Collect neurospheres on the second or third day after the passage and plate around 50 to 100 spheres into each well.

 After 48 h, fix the cells by 4% paraformaldehyde.

 Capture cell images and measure the distance between the migrated cell and the edge of aggregated neurosphere cells (Fig. 1).

 Prepare cover glasses by coating with 0.01% polyethylenimine and 5 μg/mL laminin as described above. Extensively wash the cover glasses for both sides by soaking them in PBS.

 Collect neurospheres as described above and culture them in the well containing Neurobasal/B27 medium without mitogens.

 At various days for in vitro culture, fix the cells by 4% paraformaldehyde for staining neural cell markers.

 After wash the cells with PBS, treat them with 0.25% Triton-X 100 for 10 min and change buffer to 1% normal goat serum.

 Incubate cells with primary antibodies for neural cell markers to judge differentiation. For example, neural stem cells (nestin), neurons (β-III tubulin), astrocytes (GFAP) and oligodendrocytes (CNPase) are good markers.

 After washing, react with fluorescence-labeled secondary antibody, followed by Hoechst staining for 5 min.

 Mount cover glasses with anti-quenching reagent on slide glasses.

 Under fluorescence microscope, capture images for different colors and count for neurons and glial cells from each neurosphere to calculate differentiation rate to neurons or glial cells. (Fig. 2)