Twenty μg of total protein
(determined with the DC Protein Assay, BioRad) were separated in 10% SDS- poly-acrylamide gels. The proteins were subsequently transferred to nitrocellulose membranes and hybridised with primary antibodies diluted accordingly: βIII-tubulin (ab18207) 1:5000, nestin (ab6142) 1:200 and GFAP (ab7260) 1:1000 (all from Abcam) and β-actin (sc-1616) 1:5000 (Santa Cruz). Horse radish peroxidase-conjugated anti-rabbit IgG (NA934 V) 1:3000 and anti-mouse Ion Channel Ligand Library purchase IgG (NA931 V) 1:3000 (Amersham) and anti-goat IgG (sc-2020) 1:3000 (Santa Cruz) were used as secondary antibodies. Densitometric analysis of visual blots was performed using Image Gauge 3.46 program (Fujifilm Co. Ltd.). The data were analysed using one-way ANOVA followed by Tukey’s Multiple Comparison Test (Fig. 2a–c) or by Student’s t-test ( Fig. 3) (GraphPad Prism 5.0). Cells grown in complete DMEM for 3 days (treatment 1 in Table 1) remained their native, neural stem cell state. Only one morphological phenotype with no visual outgrowth of neurites was observed in the cultures (Fig. 1a). For cells grown in conditioned complete DMEM for 8 days (treatment 2 in Table 1) or with medium change after day 4 (treatment 3 in Table 1) no morphological signs of neuronal differentiation were observed (not shown). Cells cultured
for 7 days in complete DMEM without FCS but with neurotrophic factors added (treatment 4–6 in Table 1) displayed similar phenotypes of neurons and astrocytes as cells cultured Selleckchem Ku 0059436 in DMEM:F12 medium with N2 supplements and neurotrophic factors (treatment 7–9 in Table 1). The cultures displayed two distinct layers of cells with different morphology (Fig. 1c). To further elucidate the progress of morphological differentiation, cells were also examined after 3, 7 and 10 days in DMEM:F12 medium with N2 supplements, NGF and BDNF
with a medium change every 4th day. After 3 days in this medium, some cells had formed neurites and changed their morphology to a dense cell body (Fig. 1b), as compared to most of the cells in the culture, but Astemizole also as compared to the undifferentiated progenitor control cells (Fig. 1a). After 7 days in DMEM:F12 medium with N2 supplements, NGF and BDNF, the cells were no longer in one layer but in two layers, apparently with one neuronal-like cell type growing on top of the other cell type with a distinctly different cell morphology (Fig. 1c). After 10 days in differentiation medium, a fine network of neurites and dense, rounded cell bodies were formed on top of the other cell type (Fig. 1d). The mRNA levels of the neural progenitor cell marker nestin (Fig. 2a) were attenuated after all exposure scenarios (treatments 2–9 in Table 1), as compared to control levels, (treatment 1 in Table 1), indicating maturation and differentiation of the C17.2 neural stem cells. The difference in nestin expression between the differentiation scenarios was however not significant. The mRNA levels of the neuronal biomarker, βIII-tubulin (Fig.