Isolation of Growth Cones from Mouse Brain
从小鼠脑部分离神经元生长锥
发布: 2013年08月05日第3卷第15期 DOI: 10.21769/BioProtoc.853 浏览次数: 11401
评审: Xuecai Ge
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
Growth cones are motile structures at the tips of growing neurites, which play an essential role in regulation of growth and navigation of growing axons and dendrites of neurons in the developing nervous system. This protocol describes isolation of growth cones from the brain tissue from young mice. Growth cones isolated using this protocol have been extensively characterized using electron microscopy (Pfenninger et al., 1983) and may be used for any kind of subsequent biochemical and/or functional analyses, including Western blot analysis of protein expression (Westphal et al., 2010), analysis of the activity of growth cone-accumulated enzymes (Leshchyns’ka et al., 2003; Li et al., 2013), and analysis of the endocytosis and exocytosis rates (Chernyshova et al., 2011).
Keywords: Neurons (神经元)
Growth cone (生长锥)
Neurite outgrowth (轴突生长)
Differential centrifugation (差速离心)
Biochemical analysis (生化分析)
Materials and Reagents
- Mouse brains extracted from 1-3 days old mice, frozen in liquid nitrogen and kept at -80 °C (for up to 1 year)
- Sucrose
- Purified (e.g. using Milli-Q system from Millipore) water kept at 4 °C
- Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science, catalog number: 05892791001 )
- PMSF (Sigma-Aldrich, catalog number: P7626 )
- Ethanol
- 80% sucrose (see Recipes)
- Homogenization buffer (see Recipes)
- 0.75 M sucrose buffer (see Recipes)
- 1 M sucrose buffer (see Recipes)
- 2.33 M sucrose buffer (see Recipes)
Equipment
- Potter homogenizer (Thermo Fisher Scientific, catalog number: 08-414-14A )
- 1 ml plastic pipette (Sarstedt, model: 86.1180 )
- Bench top centrifuge with an angle rotor, for example Allegra X-15R (Beckman Coulter)
- Ultracentrifuge with a swing rotor, for example L-60 ultracentrifuge with SW40Ti rotor (Beckman) or HIMAC CP100WX ultracentrifuge with P40ST rotor (Hitachi)
- Centrifuge tube 13PA (Hitachi, catalog number: 332901A )
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Leshchyns’ka, I. and Sytnyk, V. (2013). Isolation of Growth Cones from Mouse Brain. Bio-protocol 3(15): e853. DOI: 10.21769/BioProtoc.853.
- Li, S., Leshchyns'ka, I., Chernyshova, Y., Schachner, M. and Sytnyk, V. (2013). The neural cell adhesion molecule (NCAM) associates with and signals through p21-activated kinase 1 (Pak1). J Neurosci 33(2): 790-803.
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分类
神经科学 > 发育 > 神经元
细胞生物学 > 细胞器分离 > 分级分离
细胞生物学 > 组织分析 > 组织分离
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