Basic fibroblastic growth factor

Cells were maintained in stem cell media consisting of DMEM/F12 basal media, N2, and B27 supplements (Invitrogen), 20 ng/mL human recombinant epidermal growth factor, and 20 ng/mL basic fibroblastic growth factor (PeproTech Inc., Rocky Hill, NJ). For the tumorsphere formation assay, cells were plated at a density of 200 cells/well in 24-well ultra-low-attachment plates or at a density of one cell/well in 96-well plates and maintained in stem cell medium. Tumorspheres that arose within 2 weeks were recorded. For serial tumorsphere formation assays, the spheres were harvested, disaggregated with 0.25% trypsin/EDTA, filtered through a 40-μm mesh, and replated as described above. For each cell type, triplicate samples were used, and the spheres were counted by two individuals in a blind fashion.

Cells were maintained in stem cell media consisting of DMEM/F12 basal media, N2 and B27 supplements (Invitrogen), 20 ng/ml human recombinant epidermal growth factor and 20 ng/ml basic fibroblastic growth factor (PeproTech Inc., Rocky Hill, NJ, USA). For the tumorsphere-formation assay, cells were plated at a density of 200 cells/well on 24-well ultra-low attachment plates or at a density of 1 cell/well on 96-well plates and maintained in stem cell medium. Tumorspheres that arose within 2 weeks were recorded. For serial tumorsphere-formation assays, the spheres were harvested, disaggregated with 0.25% trypsin/EDTA, filtered through a 40 μm mesh and replated as described above. For each cell type, the experiments were performed in triplicate, and the spheres were counted by two individuals in a blinded fashion.

Neural progenitors were propagated from the wall of lateral ventricles of wild-type and Fmr1 KO pups as previously described (Castrén et al., 2005 (link)). Cells were grown as free-floating aggregates referred to as neurospheres in Dulbecco’s modified Eagle’s medium F-12 nutrient mixture (DMEM/F-12) media containing B27 supplement (both from Gibco, Life Technologies Ltd.), L-glutamine (2 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 15 mM), penicillin (100 U/ml), and streptomycin (100 U/ml) (all from Sigma-Aldrich), in the presence of basic fibroblastic growth factor (10 ng/ml) and epidermal growth factor (20 ng/ml) (both from PeproTech) in a 5% CO₂-humidified incubator at +37^oC. The culture medium was refreshed and growth factors were added three times per week. The cells were passaged by manual trituration at approximately two weeks intervals. Neuronal progenitor cells from WT and Fmr1 KO mice were plated at a concentration of 100000 cells/10 ml plate and grown as neurospheres for 5 days. Medium was changed and growth factors last added 5 h prior to the start of treatments. Cells were treated with 1 μM fluoxetine in parallel with corresponding non-treated controls for 48 h. The cells were then collected as cell pellets and stored at –70°C until further use.