| 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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| Extraction and Profiling of Plant Polar Glycerol Lipids
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Author:
Date:
2016-06-20
[Abstract] This protocol describes a method to extract total polar glycerol lipids from plant materials, followed by mass spectrometry profiling. Different glycerol lipid classes can be distinguished by their head-groups, which can be profiled automatically and quantitatively by a triple quadrupole mass spectrometry in multiple reaction monitoring (MRM) mode with an autosampler. Comparing with other established methods, such as thin layer chromatography (TLC) separation followed by Gas spectrometry (GC) analysis, this method requires little effort in sample preparation and separation, while the resolution is not limited to general lipid classes but at side chain level. This method was described and used successfully to profile plant lipids changes under freezing stress in Welti et al. ...
[摘要] 该方案描述了从植物材料提取总极性甘油脂质的方法，随后是质谱分析。 不同的甘油脂类可以通过其头基来区分，其可以通过使用自动进样器的多重反应监测（MRM）模式中的三重四极杆质谱自动和定量地进行概况分析。 与其他已建立的方法，例如薄层色谱（TLC）分离，然后气体光谱（GC）分析相比，该方法在样品制备和分离中几乎不需要付出努力，而分辨率不限于一般脂质类别，而是侧链水平 。 在Welti等人（2002）中描述并且成功地使用该方法来分析在冰冻胁迫下植物脂质的变化。
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