Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex
Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex
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DOI：
10.1038/nprot.2012.099
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发表时间：
2012-09-01
期刊：
影响因子：
14.8
通讯作者：
Arikkath, Jyothi
中科院分区：
文献类型：
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作者：
Beaudoin, Gerard M. J., III;Lee, Seung-Hye;Arikkath, Jyothi
培养和维持出生后小鼠海马和皮质神经元的能力是非常有利的，特别是对于基因工程小鼠模型的研究。本研究采用分离培养的方法，从出生后早期（P0-P1）的小鼠海马和皮层分离培养锥体神经元。这些低密度解离培养物在没有饲养层的聚-L-赖氨酸涂覆的玻璃基底上生长。培养的神经元存活良好，发育广泛的轴突和树突乔木，表达神经元和突触标记，并形成功能性突触连接。此外，它们非常适合低效率和高效率转染和延时成像。这种优化的细胞培养技术可用于培养和维持神经元，用于各种应用，包括免疫细胞化学、生物化学研究、shRNA介导的敲除和活体成像研究。玻璃基质的制备必须在培养前开始5 d开始。神经元的解剖和铺板需要3-4小时，神经元可以在培养物中维持长达4周。
The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.