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N-2 Supplement (100X), Liquid

N-2补充物(100X)

Company: Thermo Fisher Scientific
Catalog#: 17502048
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FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand
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; ...

Obtaining Multi-electrode Array Recordings from Human Induced Pluripotent Stem Cell–Derived Neurons
Author:
Date:
2017-11-20
[Abstract]  Neuronal electrical properties are often aberrant in neurological disorders. Human induced pluripotent stem cells (hiPSCs)-derived neurons represent a useful platform for neurological disease modeling, drug discovery and toxicity screening in vitro. Multi-electrode array (MEA) systems offer a non-invasive and label-free platform to record neuronal evoked-responses concurrently from multiple electrodes. To better detect the neural network changes, we used the Axion Maestro MEA platform to assess neuronal activity and bursting behaviors in hiPSC-derived neuronal cultures. Here we describe the detailed protocol for neuronal culture preparation, MEA recording, and data analysis, which we hope will benefit other researchers in the field. [摘要]  神经元电特性在神经疾病中经常是异常的。 人诱导的多能干细胞(hiPSC)衍生的神经元代表神经疾病建模,药物发现和体外毒性筛选的有用平台。 多电极阵列(MEA)系统提供了一个非侵入性的无标记平台,可同时记录来自多个电极的神经元诱发反应。 为了更好地检测神经网络变化,我们使用了Axion Maestro MEA平台来评估hiPSC衍生的神经元培养物中的神经元活动和爆裂行为。 在这里,我们描述了神经元培养准备,MEA记录和数据分析的详细方案,我们希望这将有益于该领域的其他研究人员。
【背景】人类诱导多能干细胞(hiPSC)技术目前正用于体外模拟神经和精神疾病。最近的研究已经证明,与特定疾病有关的某些细胞表型可以在盘中重现。神经电活动是神经系统功能的本质,代表了正常功能对于情绪,记忆,感觉形态和体内行为至关重要的交流的关键形式。在疾病状况下,电特性可能受到影响,因此了解基于hiPSC的神经疾病模型中的神经元电路连接性,生理学和病理学非常重要。

膜片钳和多电极阵列(MEA)技术是用于评估电生理学活性并由此评估神经元功能的主要技术。尽管膜片钳是研究单个细胞的活性和功能的强大的细胞内方法(Neher等人,1978),MEA平板具有记录细胞外动作电位(或尖峰)的能力,和同一板中数千个不同细胞同时的局部场电位,从而更好地理解网络水平的神经元活动(Hutzler等人,2006; ...

Isolation of Mouse Cardiac Neural Crest Cells and Their Differentiation into Smooth Muscle Cells
Author:
Date:
2017-09-05
[Abstract]  Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3rd somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells. This protocol was adapted from (Newgreen and Murphy, 2000; Pfaltzgraff et al., 2012). [摘要]  心脏神经嵴细胞(CNCC)起源于神经管的背部边缘,位于第3个体节的耳穴和尾缘之间,并迁移到咽弓和心脏。 我们已经表明,纤连蛋白(Fn1)通过调节CNCCs到咽弓动脉周围的血管平滑肌细胞的分化,在CNCC的发展中起重要作用(Wang and Astrof,2016)。 该方案描述了CNCC与神经管和尾尾弓的分离,以及神经嵴衍生细胞分化成平滑肌细胞。 该方案从(Newgreen和Murphy,2000; Pfaltzgraff等人,2012)改编。
【背景】以前发表的方案描述了从神经管分离神经嵴细胞。然而,在耳孔和第三体细胞之间的神经管区域中的神经嵴细胞包括有助于许多不同细胞类型的神经嵴细胞群体;例如,迷走神经嵴细胞也来自该区域。在该方案中,我们修改了用于分离心脏神经嵴细胞的常规方法。而不是使用神经管,我们在胚胎期(E)9.5(22-25个体节期)使用尾部咽部弓形区。这是在将心脏神经嵴细胞分化为血管平滑肌细胞之前。神经嵴培养物通常含有污染性间充质细胞,通常表达平滑肌基因。为了鉴定神经嵴衍生细胞,我们从以下交叉产生的胚胎中分离出神经嵴细胞:Fn1flox / flox; ROSAmTmG / mTmG雌性小鼠×Fn1 +/-;Tfap2αIRESCre/ ...

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