Ethylene glycol, ferric chloride, and sodium acetate were obtained from Aladdin (Shanghai, China). Sodium hydroxide and tetraethoxysilane (TEOS) were obtained from Macklin (Shanghai, China). Laminin and acrylamide were obtained from Sigma-Aldrich (MO, United States). Phosphate buffered saline (PBS), accutase, Hanks’ balanced salt solution (HBSS), B-27, and recombinant human epidermal growth factor (EGF) were obtained from Gibco (NY, United States). Recombinant murine FGF-basic (FGF) was obtained from PropTech (NJ, United States). The NeuroCult™ differentiation kit was obtained from STEMCELL Technologies (CA, United States). The nestin marker (Nestin antibody, AN205), TuJ-1 marker (neuronal class III β-tubulin, AT809), and DAPI (C1002) were obtained from Beyotime (Jiangsu, China). Donkey anti-mouse secondary antibody (A21202) was obtained from ThermoFisher (MA, United States).
Neurocult differentiation kit
Manufactured by STEMCELL
Sourced in United States
The NeuroCult™ differentiation kit is a laboratory product designed to facilitate the differentiation of neural stem/progenitor cells. The kit provides a defined medium and supplements to support the growth and differentiation of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Laminin, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Maclin Biochemical Technology (1 mentions)
Dapi c1002, by Beyotime (1 mentions)
Human recombinant epidermal growth factor egf, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Acrylamide, by Merck Group (1 mentions)
3 protocols using neurocult differentiation kit
1
Neural Stem Cell Differentiation Protocol
2
Snip1 Knockout Neural Differentiation
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For neural differentiation, NeuroCult™ Differentiation Kit (STEMCELL Technologies 05704) was used by following the manufacturer’s instructions. On Day 0, approximately 1.6 × 106 NPCs from Snip1[flox/flox] embryos were seeded onto each well of 6-well plates which were double-coated with 10 μg/mL of poly-D-lysine and 10 μg/mL laminin. On Day 1 (or Day 5), cells were incubated with either mCherry-control or mCherry-Cre lentivirus (Vector Core Lab at St. Jude Children’s Research Hospital) for 8 h, washed twice with 1X PBS, and cultured for 14 days. During culturing, a half-medium change was done when the culture media turned yellow. On Day 14, cells were harvested for RNA extraction and real-time quantitative PCR.
3
Neurosphere Differentiation and Characterization
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Primary neurospheres (described above) were mechanically dissociated by pipetting up/down 40 times and centrifuging at 300 g for 10 min at RT. The cell pellet was re-suspended in 1mL of culture media made from the NeuroCult Differentiation kit (STEMCELL Technologies 05704) and filtered through a 40 mm cell strainer. Passaged cells were counted (described above) and plated at a cell density of 5,000 cells/mL in a 24-well plate (1mL/well) with GG-12-Laminin coated glass slips (Neuvitro) at the bottom of the wells. The Laminin slip cultures were incubated for 5 days at 37 C in a 5% CO 2 atmospheric incubator, at which point 500 mL of media was added to the cultures to refresh the media. At 10 DIV, the Laminin slips were fixed with 4% PFA in preparation for IHC (see IHC protocol above).
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