Neurocult ns a differentiation kit

Self-renewability of stem cells and primary tumor cultures, in conditions with or without growth factors, was assayed as previously described (Swartling et al., 2012 (link)). Both primary and secondary spheres were allowed to form from 10 cells/well in ultra-low attachment 96-well plates in complete TC media or in TC media where EGF, bFGF, or both were removed. Seeded cells were allowed to form primary spheres for seven days, after which spheres were dissociated as described in tumor cell culture establishment section. These cells were then re-seeded (10 cells/well in ultra-low attachment plates) and allowed to grow for another seven days to form secondary spheres. Secondary spheres having diameter larger than 10, 20, 40 and 80μm were counted and statistical analysis was performed on three replicates. Sphere forming assays were done in two biological replicates.
Differentiation experiments were done by culturing stem cells and primary tumor cultures for four weeks using STEMCELL Technologies NeuroCult NS-A Differentiation Kit (Human), given new media weekly or split when needed, after which they were seeded on PO (1:500)/laminin (1:1000) coated chamber slides and cultured for an additional four days then fixed and processed for immunofluorescence. Undifferentiated cells were seeded on coated chamber slides two days before fixation.

The hPB-MSCs were seeded and incubated in the α-MEM complete medium until a confluence of about 30% was reached. The cells were exposed to neurogenic induction media (5752; NeuroCult™ NS-A Differentiation Kit, Stemcell Technologies, Canada), and the induction medium was refreshed every 2 days. The cells cultured in a basal medium of α-MEM supplemented with 10% FBS served as a negative control. The induction process lasted 3 weeks. Neurogenic differentiation was confirmed by immunofluorescence assessment of Glial Fibrillary Acidic Protein (GFAP; G3893; Sigma-Aldrich).