The differentiating neurospheres with the paradigm described above were divided into two groups 18 hrs after initial plating (to allow attachment) — receiving no additions (Differentiating Control group) or alcohol (ALC, 400mg/dL for 6 hrs; Differentiating ALC group). A study with 5-aza-cytidine (AZA, DNA-methylation inhibitor, 50ng/mL, Sigma, St. Louis, MO; Differentiating AZA group) treatment was done previously (Singh et al., 2009b). At the end of the differentiation period, the undifferentiated and differentiating cells were (a) collected at 2 DIC by scraping all the cells from the Petri dish for MeDIP analysis, or (b) washed with 0.1M PBS at 2 or 4 DIC and fixed with the 4% formaldehyde fixative in a chamber slide for immunocytochemical analysis of phenotypes.
