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Corning® 100 mL Penicillin-Streptomycin Solution, 100x

Company: Corning
Catalog#: 30-002-CI
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HIVGKO: A Tool to Assess HIV-1 Latency Reversal Agents in Human Primary CD4+ T Cells
Author:
Date:
2018-10-20
[Abstract]  While able to suppress HIV replication in HIV infected individuals, combination antiretroviral therapy (ART) fails to eliminate viral latent reservoir, which consists in integrated transcriptional silenced HIV provirus. So far, identification of latently-infected cells has relied on activating cells to induce expression of HIV proteins which can then be detected. Unfortunately, this activation significantly changed the cell phenotype. We developed a novel HIV reporter, named HIVGKO, that allows the purification of latently-infected cells in absence of reactivation. Indeed, latent cells can be identified by expression of the EF1a-driven mKO2 and lack of expression of the LTR-driven csGFP. This protocol can be used to study the effectiveness of LRAs (Latency Reversal Agents) in ... [摘要]  虽然能够抑制HIV感染个体中的HIV复制,但联合抗逆转录病毒疗法(ART)无法消除病毒潜伏性储库,其包含整合的转录沉默的HIV原病毒。 到目前为止,潜伏感染细胞的鉴定依赖于激活细胞以诱导HIV蛋白的表达,然后可以检测到这些蛋白的表达。 不幸的是,这种激活显着改变了细胞表型。 我们开发了一种名为HIV GKO 的新型HIV报告基因,可以在没有再激活的情况下纯化潜伏感染的细胞。 实际上,可以通过EF1a驱动的mKO2的表达和LTR驱动的csGFP的缺乏表达来鉴定潜伏细胞。 该方案可用于研究LCA(潜伏期逆转剂)在原代细胞中重新激活潜伏HIV的有效性。

【背景】新版双标记病毒(HIV GKO )含有5'LTR中HIV-1启动子控制下的密码子转换eGFP(csGFP)和一种独特的无关荧光蛋白 mKO2在细胞延伸因子αα启动子(EF1α)的控制下。 当使用与遗传相关的荧光蛋白时,由于重组问题,在这些报道分子中使用不相关的荧光蛋白是很重要的。 因此,生产性感染的细胞主要是csGFP + mKO2 + (有些可能只是GFP + ),而潜伏感染的细胞是csGFP - mKO2 + 。 流式细胞仪如分拣机AriaII允许纯化纯感染人群(生产性,潜伏性和/或未感染),而分析仪LSRII允许评估HIV GKO LTR的转录激活。 感染后的时间很短。

Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells
Author:
Date:
2018-02-05
[Abstract]  The uptake and trafficking of cell surface receptors can be monitored by a technique called ‘antibody-feeding’ which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the ... [摘要]  细胞表面受体的摄取和贩卖可以通过被称为“抗体 - 进纸”技术,其在外部使用上应用的抗体来标记培养的活细胞的表面上的受体进行监测。在这里,我们适应传统的抗体喂养实验极化上皮细胞(Madin-Darby犬肾)生长在透性transwell支持。通过添加两个串联胞外Myc表位标签的SNARE蛋白突触3(Stx3)的C末端,我们提供了一种站点,其中与抗体可以结合,使我们能够用不同的顶端和基底外侧膜对细胞进行抗体 - 喂养实验。通过这个程序,我们观察到Stx3的内吞和细胞内运输。具体而言,我们评估从基底外侧膜Stx3的内在化速率和在时间和空间上使用固定在不同时间点的细胞免疫荧光显微镜随后的内吞观察路径。为此,介绍了以下可追踪单层的贩运行为:来自限制膜的靶蛋白。

【背景】SNARE蛋白突触3(Stx3)是已知的建立在极化上皮细胞顶端 - 基底极性(低等人,1996年Delgrossi 等人,1997年Weimbs 等人,1997;低等人,1998;黎等人,2002;低等人

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一个典型的12孔细胞培养皿的阱内的transwell小室的聚碳酸酯膜的细胞培养插入物的图1示意图。斯特朗细胞是在膜的顶部和培养将形成紧密的单层做密封关聚碳酸酯膜将顶端介质隔室与基底外侧介质隔室分开。 ...

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