E10, E11, E12 and E13 cells were grown in the presence of EGF and FGF2, dissociated into single cells, attached to Petri dishes and allowed to differentiate for 5 days without growth factors. Each plate was stained for a different cell marker; β III tubulin for neurons, GFAP for astrocytes and O4 for oligodendrocytes. The number of differentiated cells on each plate was blind counted as percentage out of the total number of cells counted on that plate, marked by DAPI nuclear stain. The sum of the percentages from each plate was used to calculate the total differentiation rate, and the presented data are percentage out of the differentiated cells. Data are mean of 3–5 plates. As derivation day progressed, fewer neurons were formed and more astrocytes emerged. Beta3T, β III tubulin; GFAP, glial fibrillary acidic protein.
Every embryonic day (E) represents one derivation, 3–5 Petri dishes per derivation.