The differentiating neurospheres, with the paradigm described above, were divided into two groups— 5-aza-cytidine (AZA, 50ng/mL, Sigma, St. Louis, MO) (n=16 wells) or medium vehicle only (Control, n=16 wells). Control and AZA treatments were conducted simultaneously, but each were separated to their own 16-well chamber slides to avoid cross-transfer between treatment and control wells. AZA was administered at the beginning of differentiation; no additional treatment or medium changes were provided during the 4 day differentiation period. At the end of the differentiation period, the cells were washed with 0.1M PBS and fixed with the same fixative above for immunocytochemical analyses.
