Quantitation of the effect of alcohol and 5-AZA on migration of differentiated NSCs was conducted in comparison to Controls. Fluorescent or bright-field images taken at 10x magnification were overlayed with a 1×1-inch grid using Adobe Photoshop software (Adobe Systems, Boston, MA). The neurosphere was divided into three regions: core, corresponding to the densely packed inner mass of tightly clustered cells; periphery, a zone stretching out approximately 100μm from the edge of the core, consisting of early differentiating cells that were less densely packed, starting to leave the neurosphere, changing morphologically from round neuroepithelial cells to angular shaped neural-like cells, and some at the outer edge of the periphery with extended neurites; and the migrated zone, consisting of all cells beyond the periphery, which have varying degrees of differentiation. The cells in the migrated zone were counted in 8 control-differentiated and 10 alcohol-treated-differentiated neurospheres. Positive cells were stained with 4', DAPI or had a clearly distinguishable cytoplasm in brightfield from their nearest neighbor. Tightly clustered or multi-layered areas were excluded from analysis. Statistical analyses, T-tests, were done using StatView (SAS, Carey, NC).
