To exit the cell cycle for all neurons at the same time point, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 10 μM, Sigma) was added 3 times to the described cultures, every other day after final plating3536. After final plating cells were grown in N2B27 medium supplemented with 1 mM dibutyryl cAMP (Sigma) from final plating onwards and furthermore supplemented with 10 ng/ml brain derived neurotrophic factor (BDNF) and 10 ng/ml glial derived neurotrophic factor (GDNF, both R&D Systems) after incubation with DAPT.
