室管膜下区(SVZ)源祖细胞生长为神经球的初代培养

Julien  Vernerey; Karine  Magalon; Pascale  Durbec

doi:10.21769/BioProtoc.868

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Peer-reviewed

Primary Culture of SVZ-derived Progenitors Grown as Neurospheres

室管膜下区(SVZ)源祖细胞生长为神经球的初代培养

JV Julien Vernerey

KM Karine Magalon

PD Pascale Durbec email

发布: 2013年08月20日第3卷第16期 DOI: 10.21769/BioProtoc.868 浏览次数: 12199

评审: Xuecai Ge

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参见作者原研究论文

The authors used this protocol in:

Cover of The Journal of Neuroscience, featuring study using the protocol.

Feb 2013

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Abstract

SVZ-derived progenitors grown as neurospheres is a well-known model to study neural stem cell and progenitor functions such as proliferation, differentiation/self-renewal, and migration (Durbec and Rougon, 2001). This protocol is for preparing a culture of SVZ-derived progenitors from 8 early postnatal mouse brains (P0 to P3). One week after cell plating, we can observe round floating neurospheres, each resulting from the clonal expansion of a single EGF/FGF responsive neural progenitor.

Keywords: Nervous system (神经系统)

Adult brain (成人的大脑)

Stem cells (干细胞)

Subventricular zone (室管膜下区)

Primary culture (原代培养)

Materials and Reagents

New born mice
Phosphate Buffered Saline (PBS) (Life Technologies, catalog number: 14040-091 )
Hank's Balanced Salt Solution (HBSS) (Life Technologies, catalog number: 14170-088 )
Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, catalog number: 61965-026 )
Ham’s F-12 nutrient mix (F12) (Life Technologies, catalog number: 31765-027 )
Trypsin (Sigma-Aldrich, catalog number: T5266 )
Insulin (Sigma-Aldrich, catalog number: I1882 )
Holo-transferrin (Sigma-Aldrich, catalog number: T0665 )
Putrescine (Sigma-Aldrich, catalog number: P5780 )
Progesterone (Sigma-Aldrich, catalog number: P8783 )
Sodium selenite (Selenium) (Sigma-Aldrich, catalog number: S5261 )
Penicillin-streptomycin (Life Technologies, catalog number: 15140-130 )
Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10106-169 )
B27 (Life Technologies, catalog number: 17504-044 )
rhFGFbasic (Peprotech, catalog number: 167 100-18B-B )
rhEGF (Peprotech, catalog number: 167 AF-100-15A )
Neurospheres defined medium (see Recipes)
Trypsin (see Recipes)

Equipment

Sterilin Universal Container (Thermo Fisher Scientific, catalog number: 128A/P )
Standard TC BD Falcon 60 mm cell culture dish (BD Biosciences, Falcon^®, catalog number: 353002 )
Vibratom (Microm, model: HM450 )
Stereoscopic Microscope
Cell culture Hood and Incubator
Scissors, fine forceps, spatula, micro knives
Ice bucket
Fire-polished glass Pasteur pipettes
Water bath

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Vernerey, J., Magalon, K. and Durbec, P. (2013). Primary Culture of SVZ-derived Progenitors Grown as Neurospheres. Bio-protocol 3(16): e868. DOI: 10.21769/BioProtoc.868.
Vernerey, J., Macchi, M., Magalon, K., Cayre, M. and Durbec, P. (2013). Ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain. J Neurosci 33(7): 3240-3250.