In addition, the neural stem cell marker Sox-2 [25] was detected (Figure 3(b)). We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days. First, the neurosphere culture formation is highly sensitive to the procedure, as the creation is contingent on the system used to create the culture. Neurons (green) and glia (red) differentiated from human neural stem cells in culture. [7], Learn how and when to remove this template message, http://www.med.nyu.edu/fishelllab/pdfs/review9.pdf, Neural Stem Cell Culture: Neurosphere generation, microscopical analysis and cryopreservation, https://en.wikipedia.org/w/index.php?title=Neurosphere&oldid=951287008, Articles needing additional references from April 2012, All articles needing additional references, Creative Commons Attribution-ShareAlike License, A Network Protocol for generating neurospheres has been published on the, Reusable 3D cell culture tool used for growing, This page was last edited on 16 April 2020, at 12:19. The neurosphere assay is a method for studying possible proliferation and self-renewal capacity of neural stem cells, especially if the region of interest could have a silent stem cell niche in vivo [5, 11, 12, 35, 36]. For immunocytochemistry, preparations were incubated with the following primary antibodies at 5°C for 12 h in 1% BSA solution and 0.1 M PBS buffer: mouse monoclonal against Atoh1 (1 : 1000; Ab27667, Abcam®), mouse monoclonal against BrdU (5-bromo-2-deoxyuridine) (1 : 600; #05-633, Millipore®), mouse monoclonal against β-tubulin (1 : 1000; #TS293, Sigma-Aldrich®), mouse monoclonal against β-III-tubulin (1 : 1000; #Ab7751, Abcam®), rabbit polyclonal against β-III-tubulin (1 : 2000; #Ab18207, Abcam®), rabbit polyclonal against doublecortin (DCX) (1 : 1000; #Ab18723, Abcam®), mouse monoclonal against glial fibrillary acidic protein (GFAP) (1 : 1000; #MAB360, Millipore®), rabbit polyclonal against myelin basic protein (MBP) (1 : 800; #M3821, Sigma-Aldrich®), mouse monoclonal against Nestin (1 : 800; #MAB353, Millipore®), and rabbit polyclonal against Sox-2 (1 : 2000; #Ab97959, Abcam®). In addition to this essential criterion, it was possible to show the formation of neural progenitor cells both within these neurospheres and from dissociated individual cells using established immunocytochemical markers. Sign up here as a reviewer to help fast-track new submissions. These morphological and immunocytological characteristics could also be demonstrated in comparable experiments with NSCs [12, 26]. The creation of the neurosphere assay is highly sensitive; it is still unclear as to the exact differing effects that environment produces, relative to the in vivo environment. To see the entire protocol, click on the source link. This site uses cookies. The cells were not immunoreactive for neurofilament, neuron-specific enolase (NSE), and glial fibrillary acidic protein (GFAP). The cardinal characteristics of neural stem cells—the ability to undergo mitotic division for unlimited self-renewal, the formation of neural progenitor cells, and the multipotency to mature into all cell types of the neuroectodermal line [11, 12, 26]—were demonstrated in this series of experiments.
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