Three MAPK inhibitor different differentiation medium compositions were used; (1) complete DMEM, (2) complete DMEM without FCS but supplemented with NGF and BDNF
[10 ng/ml of each neurotrophic factor], and (3) DMEM:F12 medium with N2 supplements (Bottenstein and Sato, 1979) together with NGF and BDNF (RnD systems Inc.). Along with the three different media, three different exposure conditions were studied; conditioned medium (no change of differentiation medium for 7 days), exchange of the differentiation medium every 3rd day and conditioned differentiation medium with addition of NGF and BDNF to the media every 3rd day. The differentiation conditions are summarised in Table 1. To morphologically characterise the differentiation process, 2.15 × 103 cells were seeded in a 8 cm2 cell culture plate in complete DMEM one day prior medium change. The cells
undergoing differentiation were treated for 7 days. Native neural stem cells kept in complete DMEM for 3 days were used as control cells. In addition to the nine exposure scenarios described above and in Table 1 for 7 days, the morphological differentiation process was followed in more detail at day 3, 7 and 10 by culturing the cells in DMEM:F12 medium with N2 supplements, NGF and BDNF [10 ng/ml] with a change of the medium every 3rd day. For analysis with reverse transcriptase (RT)-polymerase chain reaction (PCR), 1.9 × 104 cells were seeded in an 8 cm2 cell culture plate in complete DMEM one day prior medium was changed to the differentiation media. Cells Talazoparib order were lysed and mRNA was isolated using the Qiagen RNeasy kit (Fermenta) after 7 days of exposure for the differentiation conditions (Table 1). Native cells kept in complete DMEM medium for three days were used as the neural stem cell control. Two Orotidine 5′-phosphate decarboxylase μg of RNA was reversed transcribed to yield cDNA by the use of specific primers. The following primer sequences were used; nestin 5‘-GGAGGGCAGAGAAGACAGTG-3‘ and 5‘-TGACATCCTGGACCTTGACA-3‘, βIII-tubulin 5‘-GAATGACCTGGTGTCCGAGT-3‘ and 5‘-CAGAGCCAAGTGGACTCACA-3‘ and GFAP 5‘-CACGAACGAGTCCCTAGAGC-3‘ and 5‘-TCACATCACCACGTCCTTGT-3‘ and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference;
5‘-GGCATTGCTCTCAATGACAA-3‘ and 5‘-TGTGAGGGAGATGCTCAGTG-3‘. The mRNA levels of nestin, βIII-tubulin and GFAP were analysed after 22–26 PCR cycles. The PCR products were analysed on 1.5% agarose gels and visualised with ethidium bromide and UV radiation. The intensity of the bands was quantified with the Image Gauge 3.46 program (Fujifilm Co. Ltd.). Based on the results from the morphological evaluation and mRNA expression, the protein expression levels after differentiation were studied. 1.9 × 104 cells were seeded in a 55 cm2 cell culture plate in complete DMEM one day prior differentiation. Differentiation proceeded in DMEM:F12 with N2 supplements, NGF and BDNF [10 ng/ml of each neurotrophic factor] (treatment 8 in Table 1) followed by Western blot analysis.