Isolation and Culture of Hippocampal Neurons from Prenatal Mice
Isolation and Culture of Hippocampal Neurons from Prenatal Mice
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DOI：
10.3791/3634
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发表时间：
2012-07-01
影响因子：
1.2
通讯作者：
Wooten, Marie W.
中科院分区：
文献类型：
--
作者：
Seibenhener, Michael L.;Wooten, Marie W.
大鼠和小鼠海马神经元的原代培养被广泛用于揭示神经生物学中的细胞机制。通过分离和培养单个神经元，研究人员能够使用各种生化技术分析与细胞运输、细胞结构和单个蛋白质定位相关的特性。这类实验的结果对于测试涉及记忆和学习的神经基础的理论至关重要。然而，这些形式的实验得出的明确结果是基于培养神经元的能力，并将其他脑细胞类型的污染降至最低。在该方案中，我们使用专为神经元生长和仔细解剖胚胎海马区组织而设计的特定培养液，以优化健康神经元的生长，同时将污染细胞类型(即星形胶质细胞)降至最低。由于解剖样本的大小，胚胎小鼠的海马体组织可能比类似的啮齿动物组织更难分离。我们展示了胚胎第19天(E19)小鼠海马体的详细解剖技术。一旦分离了海马区组织，就可以通过稀释浓度的胰酶和机械破坏来实现神经细胞的温和分离，以将细胞与结缔组织分离，同时对单个细胞造成最小的损害。详细描述了如何准备用于中断的移液管。为免疫荧光法提供了最佳的培养密度，以最大限度地提高细胞培养的成功率。该方案为从小鼠海马区组织中培养神经细胞提供了一种快速(约2小时)和有效的技术。
Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.