The neurospheres at passages four to five were used for this analysis. Five to 10 neurospheres were allowed to adhere to coverslips coated with poly-l-ornithine and fibronectin in a 24-well plate containing neural culture medium, supplemented with 2% (v/v) B27. The cells were cultured in an atmosphere containing 4% oxygen and 37% carbon dioxide for 48 h. After culturing, the cells were stained with the neurite marker, βIII-tubulin and the neuronal nuclear marker, NeuN. Average neurite lengths and migration distances from each neurosphere were measured using NIH Image J.
