Kaech Stefanie, Banker Gary
Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, L606, 3181 SW Sam Jackson Park Rd., Portland, Oregon 97239, USA.
Nat Protoc. 2006;1(5):2406-15. doi: 10.1038/nprot.2006.356. Epub 2007 Jan 11.
We provide protocols for preparing low-density dissociated-cell cultures of hippocampal neurons from embryonic rats or mice. The neurons are cultured on polylysine-treated coverslips, which are suspended above an astrocyte feeder layer and maintained in serum-free medium. When cultured according to this protocol, hippocampal neurons become appropriately polarized, develop extensive axonal and dendritic arbors and form numerous, functional synaptic connections with one another. Hippocampal cultures have been used widely for visualizing the subcellular localization of endogenous or expressed proteins, for imaging protein trafficking and for defining the molecular mechanisms underlying the development of neuronal polarity, dendritic growth and synapse formation. Preparation of glial feeder cultures must begin 2 weeks in advance, and it takes 5 d to prepare coverslips as a substrate for neuronal growth. Dissecting the hippocampus and plating hippocampal neurons takes 2-3 h.
我们提供了从胚胎大鼠或小鼠制备海马神经元低密度解离细胞培养物的方案。神经元培养在经聚赖氨酸处理的盖玻片上，盖玻片悬浮在星形胶质细胞饲养层上方，并在无血清培养基中维持培养。按照此方案培养时，海马神经元会形成适当的极性，发育出广泛的轴突和树突分支，并相互形成大量功能性突触连接。海马培养物已被广泛用于可视化内源性或表达蛋白的亚细胞定位、成像蛋白运输以及确定神经元极性、树突生长和突触形成发育背后的分子机制。胶质饲养细胞培养物的制备必须提前2周开始，制备盖玻片作为神经元生长的底物需要5天时间。解剖海马体并接种海马神经元需要2 - 3小时。
