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GlutaMAXTM Supplement

GlutaMAX TM补充

Company: Thermo Fisher Scientific
Catalog#: 35050061
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Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells
Author:
Date:
2017-11-05
[Abstract]  There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation in a reductionist model. While the neurosphere assay is primarily performed to directly study the proliferative and differentiation potential of cells in individual brains, the monolayer culture allows single cell analysis in a rather homogeneous cell population. Here, we describe the isolation, culturing methods and differentiation of neural precursor cells in both systems. [摘要]  成年哺乳动物脑中有两个神经生态位:侧脑室下脑室区和海马齿状回颗粒下区。 来自这些区域的细胞可以在体外分离和维持,使用两种不同的培养系统评估它们在还原模型中的增殖和分化的潜力。 虽然神经球测定主要是为了直接研究个体脑中细胞的增殖和分化潜能,单层培养允许在相当均匀的细胞群中进行单细胞分析。 在这里,我们描述了两个系统中的神经前体细胞的分离,培养方法和分化。

【背景】在哺乳动物脑中,成人神经干细胞存在于两个主要神经生态位中,即海马齿状回(DG)的下颗粒区(SGZ)和室下区(SVZ)的侧脑室,其允许新生神经元成人的大脑。来自神经生态位的神经前体细胞可以在体外分离和培养以模拟细胞过程,尤其是增殖和分化。两种标准培养系统,贴壁单层培养(Palmer等人,1995; Ray等人,1995)和神经球测定(Reynolds和Weiss,1992和1996 )在20世纪90年代被引入,代表了在体外研究神经祖细胞生物学的有价值的工具。

根据研究问题,每个系统都有其优点和缺点,在选择其中一种或另一种培养方法之前应该仔细考虑。在贴壁单层培养中,细胞生长相当孤立,形成更均匀的培养物。单层允许直接调查和监测单细胞水平的神经前体细胞。受控条件下的形态,增殖和分化等特征可以很容易地分析和可视化。然而,与神经球培养物相比,以单层培养的细胞代表更复杂的模型,因为细胞通常以更少的通常存在于细胞壁中的细胞 ...

DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
Author:
Date:
2017-10-05
[Abstract]  Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3’-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the ... [摘要]  溶酶体是具有降解递送到这些隔室的货物的活性水解酶所需的酸性环境的内吞途径的末端。 这种内溶溶酶体的货物递送和降解过程是一个严格调节的过程,对于维持细胞体内平衡是重要的。 Cargos如EGF(表皮生长因子),Dil-LDL(3,3'-二十八碳基碳代花青 - 低密度脂蛋白),葡聚糖,DQ-BSA(染料淬灭 - 牛血清白蛋白)等常规 由研究人员用来分析各种蛋白质在内吞途径中的作用。 在缺乏或过表达感兴趣的基因的细胞中的DQ-BSA的贩运是用于鉴定各种蛋白质在内吞运输途径中的作用的有用测定。 该方案描述了可用于研究各种细胞类型的吞噬运输的DQ-Red BSA运输测定。
【背景】细胞不断地与其细胞外环境交换物质,并且在这个过程中,它们将货物内部化在质膜的囊泡中。这种内部货物被运送到早期的内体,从那里它可以通过再循环内体回到质膜或进入规范的内吞途径。一旦被注定要退化,货物就会移动到后期的内体,并最终与溶酶体融合,其中活性水解酶消化货物(Jovic等人,2010)。这些内吞室具有其内腔的特征pH。早期内体体内的pH值范围为5.9-6.8,晚期内体的pH范围为4.9-6.0,溶酶体最为酸性,pH范围为4.5-5.0(Maxfield和Yamashiro,1987)。溶酶体的酸性环境对于存在于其管腔中的水解酶和货物降解的活性是必需的(Garg等人,2011; ...

GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling
Author:
Date:
2017-09-05
[Abstract]  High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved ... [摘要]  高含量筛选是了解复杂细胞过程和鉴定调节这种途径的基因,蛋白质或小分子化合物的有用工具。高含量测定法通过显现基于图像的读数的变化来监测蛋白质或途径的功能,例如报告蛋白的定位的变化。其实例可以是将荧光标记的蛋白质从细胞质转移到细胞核或质膜。已知发生这种易位的一种蛋白质是生长因子受体结合蛋白2(GRB2),其在刺激生长因子受体并且随后经历内化后被招募到质膜。我们以前用GFP标记的Grb2来鉴定涉及EGFR信号的基因(Petschnigg等,2017)。最终,该测定可以适应于cDNA表达克隆(Freeman等人,2012),并且可用于早期药物发现以鉴定调节或抑制EGFR信号传导和内化的化合物(Antczak和Djaballah,2016)。
【背景】生长因子受体的信号转导对细胞维持正常功能至关重要,因此需要严格控制。生长因子受体的信号转导通过外部配体(例如,表皮生长因子,EGF)与跨膜受体(例如表皮生长因子受体(EGFR))和下游信号级联的活化(Yao等人,2015 )。 EGFR-信号传导的关键调节因子是生长因子受体结合蛋白2(Grb2),其由两个SH3结构域的内部SH2(Src同源性2)结构域组成。 Grb2通过其SH2结构域结合磷酸化酪氨酸残基上的活化生长因子受体,从而将受体活化与SOS-Ras-MAPK(丝裂原活化蛋白激酶)信号级联偶联。 ...

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