A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.
A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus.
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DOI：
10.3389/fnins.2011.00089
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发表时间：
2011
影响因子：
4.3
通讯作者：
Kempermann G
中科院分区：
文献类型：
--
作者：
Babu H;Claasen JH;Kannan S;Rünker AE;Palmer T;Kempermann G
体外试验是研究成体神经前体细胞在受控条件下的特性的有价值的工具，具有一组定义的参数。我们在这里提出了一个详细的协议的基础上，我们以前的原始出版物（巴布等人，）从成年小鼠海马分离神经前体细胞，并将其维持和增殖为贴壁单层培养物。该策略是基于使用Percoll密度梯度离心富集前体细胞从微解剖齿状回。基于Nestin和Sox 2的表达，可以实现大于98%的培养物纯度。在添加有丝分裂原表皮生长因子和成纤维细胞生长因子2以及补充剂Glutamax-1和B27的Neurobasal A培养基中，在无血清条件下扩增培养物。在分化条件下，前体细胞可靠地产生约30%的神经元，具有适当的形态，分子和电生理特性，可能反映颗粒细胞的特性，作为其在体内的对应物。我们还强调了对协议的潜在修改。
In vitro assays are valuable tools to study the characteristics of adult neural precursor cells under controlled conditions with a defined set of parameters. We here present a detailed protocol based on our previous original publication (Babu et al.,) to isolate neural precursor cells from the hippocampus of adult mice and maintain and propagate them as adherent monolayer cultures. The strategy is based on the use of Percoll density gradient centrifugation to enrich precursor cells from the micro-dissected dentate gyrus. Based on the expression of Nestin and Sox2, a culture-purity of more than 98% can be achieved. The cultures are expanded under serum-free conditions in Neurobasal A medium with addition of the mitogens Epidermal growth factor and Fibroblast growth factor 2 as well as the supplements Glutamax-1 and B27. Under differentiation conditions, the precursor cells reliably generate approximately 30% neurons with appropriate morphological, molecular, and electrophysiological characteristics that might reflect granule cell properties as their in vivo counterpart. We also highlight potential modifications to the protocol.
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