| FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand
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Author:
Date:
2017-12-20
[Abstract] Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and ...
[摘要] 从新鲜组织中直接分离人类神经和胶质瘤干细胞允许其在没有事先培养的情况下进行生物学研究，并且可以在其天然状态中捕获其分子表型的新方面。最近，我们展示了前瞻性地从新鲜人类生发基质和胶质母细胞瘤样品中分离干细胞群的能力，利用细胞在荧光激活细胞分选（FACS）中结合表皮生长因子（EGF）配体的能力。我们证明FACS分离的EGF结合的神经和成胶质细胞瘤细胞群体在体外包含球体形成的集落，并且能够自我更新和多向分化。在此我们详细描述了具有来自新鲜死亡和手术组织的干细胞特性的EGF-结合（即EGFR +）人类神经和胶质瘤细胞的纯化方法。利用天然配体结合能力前瞻性分离干细胞群的能力为了解非培养条件下的正常和肿瘤细胞生物学打开了新的门，并且适用于在种群和单细胞分辨率下的各种下游分子测序研究。
【背景】由于缺乏通用的神经和神经胶质瘤干细胞标志物（Lathia et al。，2015）以及频繁依赖于培养的细胞，理解人神经和胶质瘤干细胞的内在生物学一直是一个挑战比那些直接从组织分离的。跨膜糖蛋白Prominin或CD133是分离神经（Uchida等，2000）和神经胶质瘤干细胞（GSC）（Singh等，2000）的最好描述和经常使用的干细胞标记物之一。等人，2003; Singh等人，2004; ...
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| Primary Culture of Mouse Neurons from the Spinal Cord Dorsal Horn
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Author:
Date:
2017-01-05
[Abstract] Primary afferents of sensory neurons mainly terminate in the spinal cord dorsal horn, which has an important role in the integration and modulation of sensory-related signals. Primary culture of mouse spinal dorsal horn neuron (SDHN) is useful for studying signal transmission from peripheral nervous system to the brain, as well as for developing cellular disease models, such as pain and itch. Because of the specific features of SDHN, it is necessary to establish a reliable culture method that is suitable for testing neural response to various external stimuli in vitro.
[摘要] 感觉神经元的主要传入主要终止于脊髓背角，其在感觉相关信号的整合和调节中具有重要作用。小鼠脊髓背角神经元（SDHN）的原代培养可用于研究从周围神经系统到脑的信号传递，以及用于发展诸如疼痛和瘙痒的细胞疾病模型。由于SDHN的具体特征，有必要建立一种可靠的培养方法，适合于测试体外各种外部刺激的神经反应。
背景 不同于目前用于培养分离的小鼠来自海马或大脑皮质的原代神经元的方案，报道了很少的在体外培养SDHN的方法。该协议主要基于以前描述的方法（Hu et al。，2003; Hugel和Schlichter，2000）。在这里，我们进行了一些修改，包括试剂，食谱，解剖和描述从新生小鼠的初步SDHN的解剖和培养的分步程序。在该方案中，使用来自新鲜脊髓背角组织的酶（木瓜蛋白酶）消化方法直接获得神经元。体外SDHN的培养可以用于进一步的实验，例如电生理记录，免疫细胞化学和Ca 2+成像，其更好地支持脊髓中的细胞行为。
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| Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers
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Author:
Date:
2012-07-20
[Abstract] Microfluidics chamber is an ideal tool to study local events that occurring in neuronal projections by perfectly compartmentalizing the cell soma from certain branches. It is very well suited for live cell imaging or immunohistochemistry staining. This protocol has been carefully modified in detail to fit the requirement of primary rat hippocampal neuronal cultures. It can also be applied to a more general neuronal culture purpose in microfluidics.
[摘要] 微流体室是一个理想的工具，研究发生在神经元投射的本地事件，通过完美地划分细胞体细胞从某些分支。 它非常适合活细胞成像或免疫组织化学染色。 这个协议已经仔细修改，以适应初级大鼠海马神经元文化的要求。 它也可以应用于更一般的神经元文化目的在微流体。
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