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Neurocult ns a differentiation kit

Manufactured by STEMCELL
Sourced in Germany, Canada

The NeuroCult NS-A Differentiation Kit is a laboratory reagent designed for the differentiation of neural stem and progenitor cells. It provides a defined, serum-free culture system for the in vitro growth and differentiation of these cell types.

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5 protocols using neurocult ns a differentiation kit

1

Multilineage Cell Differentiation Protocol

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Cells were seeded at a density of 1×105 cells/well in 24-well plates in growth medium. At 80% confluence, the medium was replaced by each differentiation medium. Medium was changed every 3 days and the experiments were terminated at day 7 for neuron, day 14 for adipocyte, osteoblast and myocyte, and day 21 for endothelium and chondrocyte. We purchased the differentiation media for adipocytes, osteoblast and chondrocyte from PromoCell (Heidelberg, Germany), for neuron from NeuroCult NS-A differentiation Kit (StemCell, Vancouver, BC, Canada), and for epithelium from Lonza (Rockland, ME, USA). For myogenic media, we used conditioned medium obtained from human primary skeletal muscle cell culture.
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2

Neural Precursor Cell Differentiation

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Monolayers of drNPCs were plated onto adherent Corning® CellBIND® culture dishes (Corning, Product #2394 to #3296) in either maintenance culture medium (as mentioned above) or in the NeuroCult NS-A Differentiation Kit, comprising of NeuroCult XF basal medium (catalog # 05760) and NeuroCult™ NS-A Differentiation Supplement (Human) (Component# 0574) (StemCell Technologies). Media was changed after every 2–3 days until each plate was used for PCR analysis. The differentiation commenced for 10 days, and the differentiation profile was analyzed.
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3

Induction of ESSC Proliferation and Differentiation

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E18 ESSCs were cultured using the NeuroCult NS-A Proliferation Kit and NeuroCult NS-A Differentiation Kit (rat) from Stem Cell Technologies, for the induction of proliferation and differentiation, respectively. The growth factor supplements consisting of insulin-like growth factor-1 (IGF-1, Sigma I 8779), leukaemia inhibitory factor (LIF, Sigma L 5158), epidermal growth factor (EGF, Sigma E 4127) and basic fibroblast growth factor (bFGF, Sigma F 0291) were purchased from Sigma. The concentrations of these growth factors used were 20 ng/mL for the EGF and bFGF, 100 ng/mL for the IGF-1 and 20 ng/mL for the LIF, in accordance with our recently published report [7 (link)]. The ESSCs were plated in T-25 culture flasks (BD Falcon) in triplicate, under five different conditions (Figure 1): group A (no growth factor); group B (EGF + bFGF); group C (EGF + bFGF + LIF); group D (EGF + bFGF + IGF-1); and group E (EGF + bFGF + LIF + IGF-1).
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4

Stem Cell Renewal and Differentiation

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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.
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5

Neurogenic Differentiation of hPB-MSCs

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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).
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