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DMEM, high glucose, GlutaMAXTM Supplement, pyruvate

DMEM,高葡萄糖,GlutaMAXTM补充剂,丙酮酸盐

公司名称: Thermo Fisher Scientific
产品编号: 10569010
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Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line
Author:
Date:
2017-05-20
[Abstract]  There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products. [摘要]  有几个计算机程序尝试预测个体在剪接供体/受体位点的序列变异的功能影响,但实验确认是有问题的,没有携带变体的个体的RNA来源。借助于外显子捕获载体,例如pSPL3,研究人员可以通过在哺乳动物细胞中表达参考和变体小基因来测试剪接位点序列变化是否导致改变的RNA剪接,并分析所得RNA产物。

背景 我们希望通过实验测试在TEK基因中鉴定的两个剪接供体位点变体c.760 + 2T> C和c.3300 + 2delT的功能影响(Souma等人,2016)。通常情况下,携带这些序列变体的个体不能获得细胞或mRNA的样品,因此我们利用外显子捕获方法作为功能测试。来自患者的DNA样品可用于感兴趣的基因组区域的PCR扩增。如果患者gDNA样品不可用,也可以通过诸如基于PCR的定点诱变等方法将序列变体并入野生型序列。
 外显子捕获方法最初是为了鉴定长期基因组DNA中的未知外显子而开发的(Duyk等人,1990)。创建了pSPL3外显子捕获载体以提高外显子鉴定的效率和可靠性,并且还允许筛选更大的基因组片段(Church et al。,1994; Nisson等,1994)。 pSPL3载体含有由SV40启动子组成的小型人造基因,具有功能性剪接供体和受体位点的外显子 - 内含子 - ...

Macrophage Inflammatory Assay
Author:
Date:
2014-07-20
[Abstract]  Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in ... [摘要]  巨噬细胞代表广泛分布的和功能不同的先天骨髓细胞群,参与对病原体的炎症反应,组织内稳态和组织修复(Murray和Wynn,2011)。巨噬细胞可以大致分为具有相反活性的两个亚群:M1或促炎性巨噬细胞,其促进T辅助1型(Th1)细胞免疫和组织损伤,以及M2或抗炎或交替激活的巨噬细胞涉及Th2反应和分辨率的炎症。在这里我们描述了一种快速测定,我们以前用于监测由脂多糖(LPS)激活的巨噬细胞在响应于由成体干细胞产生的治疗性旁分泌因子的促炎和抗炎细胞因子产生中的变化(Bartosh等,/em>,2010; Ylostalo等人,2012; Bartosh 等人,2013)。该测定可以适当地适应于测试巨噬细胞对其它试剂的响应,在本文中将称为"测试试剂"或"测试化合物"。在该方案中,小鼠巨噬细胞细胞系J774A.1被扩增作为陪替氏培养皿上的粘附单层,允许容易地收获细胞而没有可以损伤细胞的酶或细胞刮擦器。然后用LPS悬浮刺激大孔,并接种到含有试验试剂的12孔细胞培养板中。 16-18小时后,收集由巨噬细胞调节的培养基,并用酶联免疫吸附测定(ELISA)测定培养基中的细胞因子谱。我们常规测量促炎细胞因子TNF-α和抗炎细胞因子白细胞介素-10(IL-10)的水平。

Radioactive Pulse-Chase Analysis and Immunoprecipitation
Author:
Date:
2014-04-20
[Abstract]  Labeling of newly-synthesized polypeptides with radioactive amino acids followed by immunoprecipitation allows quantitative analysis of the fate of a given protein in a time-dependent manner. This biochemical approach is usually used to study a variety of processes, such as protein folding, co-translational modifications, intracellular transport, and even its rate of degradation. Here, I describe step by step a simple technique to both label newly-synthesized influenza A virus (IAV) hemagglutinin (HA) with [35S]-methionine and then follow its maturation and transport through the ... [摘要]  用放射性氨基酸标记新合成的多肽,随后免疫沉淀允许以时间依赖性方式定量分析给定蛋白质的命运。 这种生物化学方法通常用于研究各种过程,如蛋白质折叠,共翻译修饰,细胞内转运,甚至其降解速率。 在这里,我逐步描述一个简单的技术,以标记新合成的甲型流感病毒(IAV)血凝素(HA)与[35 S] - 甲硫氨酸,然后跟随其成熟和运输通过分泌 通过SDS-PAGE和荧光成像(Magadan等人,2013)。

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