Isolation and culture of adult neurons and neurospheres
Isolation and culture of adult neurons and neurospheres
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DOI：
10.1038/nprot.2007.207
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Torricelli, John R.
中科院分区：
文献类型：
--
作者：
Brewer, Gregory J.;Torricelli, John R.
在这里，我们提出了一个协议的提取和培养神经元从成年大鼠或小鼠中枢神经系统。该方法规定了切片的优化蛋白酶消化，用Hibernate控制孵育器外的渗透压和pH值，以及神经元与碎片的密度梯度分离。该方案从每个大脑产生数百万个皮质、海马神经元或神经球祖细胞。整个神经元分离培养过程不到4 h。使用合适的生长因子，培养物中轴突和树突的成年神经元再生进行1-3周，以允许药理学、电生理学、发育、再生和神经毒理学的对照研究。成年神经球可以在1周内收集，作为在伦理上优于胚胎或胎儿来源的神经祖细胞来源。该方案强调了神经元分化和神经球增殖之间的两个差异：粘附依赖性和醋酸视黄酯的分化能力。
Here we present a protocol for extraction and culture of neurons from adult rat or mouse CNS. The method proscribes an optimized protease digestion of slices, control of osmolarity and pH outside the incubator with Hibernate and density gradient separation of neurons from debris. This protocol produces yields of millions of cortical, hippocampal neurons or neurosphere progenitors from each brain. The entire process of neuron isolation and culture takes less than 4 h. With suitable growth factors, adult neuron regeneration of axons and dendrites in culture proceeds over 1-3 weeks to allow controlled studies in pharmacology, electrophysiology, development, regeneration and neurotoxicology. Adult neurospheres can be collected in 1 week as a source of neuroprogenitors ethically preferred over embryonic or fetal sources. This protocol emphasizes two differences between neuron differentiation and neurosphere proliferation: adhesion dependence and the differentiating power of retinyl acetate.