Poly ornithine fibronectin coated 48 well culture plates

Spheres were dissociated mechanically to single-cell suspensions and replated onto poly-ornithine/fibronectin-coated 48-well culture plates (Corning, NY) at 6.5 x 10⁴ cells/cm². One hr after plating, the differentiation of NSCs was induced by replacing bFGF-containing medium with fresh N2 medium supplemented with 1% fetal bovine serum (FBS) and culturing for 3 days.

Dissociated single-cell suspensions were plated onto poly-ornithine/fibronectin-coated 48-well culture plates (Corning, NY), and were maintained to allow adhesion at 37°C in 5% CO₂/ 95% air. One hour after plating, proliferating NSCs were determined by using the Click-iT EdU Alexa Fluor 488 Imaging kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, cells were incubated with 10 μM EdU for 1h before fixation, permeabilization, and EdU staining. Cell nuclei were stained with DAPI. Immunostaining for nestin was performed as described in the Immunostaining protocol. For fetal brain-derived NSCs, 10 fluorescence images of different fields (1.3 × 1.8 mm) were obtained using a fluorescence microscope (Axio Imager. M2, Carl Zeiss, Jena, Germany). The percentages of EdU/nestin double-positive cells (% of total nestin-positive count) were then determined using ImageJ (National Institutes of Health, Bethesda, MD). For iPSC-derived NSCs, EdU/nestin double-positive cells were counted using the IN Cell Analyzer 2200 (GE Healthcare). An average of nine images per well were collected using a 10× objective. Data analysis was performed using the IN Cell Developer Toolbox software.