Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0.5) C57Bl/6J mice
Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0.5) C57Bl/6J mice
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DOI：
10.1016/j.jneumeth.2005.05.022
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发表时间：
2005-12-15
影响因子：
3
通讯作者：
Baumgart-Vogt, E
中科院分区：
文献类型：
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作者：
Ahlemeyer, B;Baumgart-Vogt, E
敲除小鼠模型允许从不同的脑区域制备原代神经元培养物，以研究与严重的、区域性不同的脑病理（例如，齐薇格综合征，过氧化物酶体生物发生障碍的最严重形式）相关的人类代谢疾病的潜在神经元病理机制。然而，患有Zellweger综合征的纯合子小鼠幼崽通常在出生后不久死亡。因此，在本研究中，我们建立了优化的方案，用于同时制备和培养来自个体新生（P0.5）C57 BI/6 J小鼠的不同脑区（内侧新皮质、海马和小脑）的无血清原代神经元培养物。对于三种类型的神经元培养物中的每一种，我们优化了分离程序和培养条件，包括包被底物、酶消化、研磨模式、接种密度和培养基组成。如通过使用抗NeuN、GFAP和CN β的抗体的间接免疫荧光所示，不同神经元培养物的纯度高。在所有神经元培养物中，少突胶质细胞的百分比小于1%。皮质、海马和小脑培养物中星形胶质细胞的比例分别为5%、7%和10%。阿糖胞苷（AraC）治疗减少的星形胶质细胞的百分比只显着在海马文化，然而，增加了海马和皮层文化中的凋亡神经元的百分比。(c)2005 Elsevier B. V.保留所有权利。
Knockout mouse models allow preparation of primary neuronal cultures from distinct brain regions in order to investigate the underlying neuronal pathomechanisms of human metabolic diseases associated with severe, regionally distinct brain pathologies (e.g. Zellweger syndrome, the most severe form of a peroxisomal biogenesis disorder). However, homozygous mouse pups with Zellweger syndrome usually die shortly after birth. Therefore, in this study, we established optimized protocols for the simultaneous preparation and cultivation of serum-free primary neuronal cultures from distinct brain regions (medial neocortex, hippocampus and cerebellum) from individual newborn (P0.5) C57BI/6J mice. For each of the three types of neuronal cultures, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, mode of trituration, seeding density and composition of the culture medium. As indicated by indirect immunofluorescence using antibodies against NeuN, GFAP and CNPase, the purity of the distinct neuronal cultures was high. The percentage of oligodendrocytes was less than 1% in all neuronal cultures. Only 5% astrocytes were present in cortical, 7% in hippocampal and 10% in cerebellar cultures. Cytosine arabinofuranoside (AraC) treatment reduced the percentage of astrocytes only significantly in hippocampal cultures, however, increased the percentage of apoptotic neurons in hippocampal and cortical cultures. (c) 2005 Elsevier B.V. All rights reserved.