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MEM Non-Essential Amino Acids Solution (100X)

MEM非必需氨基酸溶液,100X

Company: Thermo Fisher Scientific
Catalog#: 11140050
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Activation of Fibroblast Contractility via Cell-Cell Interactions and Soluble Signals
Author:
Date:
2018-09-20
[Abstract]  The collagen contraction assay is an in vitro, three-dimensional method to determine the factor(s) affecting the contractile behavior of activated cells such as fibroblasts in either physiological or pathological scenarios. The collagen lattices/hydrogels are seeded with fibroblasts to mimic the interactions between these cells and their surrounding extracellular matrix proteins in the connective tissue. This method is an important platform to assess components as potential therapeutic targets to prevent pathologies such as fibrosis, which are manifestations of hyperactivated fibroblasts. We have described a basic version of this collagen contraction assay, which is amenable to customization using different cell types under diverse experimental conditions. [摘要]  胶原收缩测定是体外三维方法,用于确定影响生理或病理场景中活化细胞如成纤维细胞的收缩行为的因子。 胶原蛋白晶格/水凝胶用成纤维细胞接种,以模拟这些细胞与其周围细胞外基质蛋白在结缔组织中的相互作用。 该方法是评估组分作为潜在治疗靶标的重要平台,以预防纤维化等病症,这些病症是过度活化的成纤维细胞的表现。 我们已经描述了这种胶原收缩测定的基本版本,其适于在不同实验条件下使用不同细胞类型进行定制。

【背景】细胞外基质的组织收缩和重塑是许多生理条件(例如伤口愈合)中的基本过程。这两种现象的核心是成纤维细胞,它不仅产生和分泌细胞外基质蛋白,而且还可以通过机械相互作用重组它们。有趣的是,这些细胞行为通常在诸如纤维化的病理条件下被夸大(Desmoulière et al。,2005),从而说明需要理解这些过程的分子调节。虽然人们早就知道,胶原蛋白是细胞外基质的主要成分之一,是组织收缩的主要参与者(Bell et al。,1979),对机械细节的透彻理解。这个过程仍然难以捉摸。对体外成纤维细胞胶原基质体外收缩的研究使研究人员能够识别导致组织收缩的新型运动员(Ngo et al。,2006; Su and Chen, 2015年)。基于该测定,可溶性因子如TGFβ(Levi-Schaffer 等,1999)和免疫细胞(Garbuzenko et al。,2002; ...

HCV Reporter System (Viral Infection-Activated Split-Intein-Mediated Reporter System) for Testing Virus Cell-to-cell Transmission ex-vivo
Author:
Date:
2018-08-05
[Abstract]  Hepatitis C virus (HCV) spread involves two distinct entry pathways: cell-free transmission and cell-to-cell transmission. Cell-to-cell transmission is not only an efficient way for viruses to spread but also an effective method for escaping neutralizing antibodies. We adapted the viral infection-activated split-intein-mediated reporter system (VISI) and developed a straightforward model for Live-cell monitoring of HCV cell-to-cell transmission ex-vivo: co-culture of HCV infected donor cells (red signal) with uninfected recipient cells (green signal) and elimination of the cell-free transmission by adding potent neutralizing antibody AR3A in the supernatant. With this model, the efficiency of cell-to-cell transmission can be evaluated by counting the number of foci designated by ... [摘要]  丙型肝炎病毒(HCV)传播涉及两种不同的进入途径:无细胞传播和细胞间传播。 细胞间传播不仅是病毒传播的有效方式,也是逃避中和抗体的有效方法。 我们采用了病毒感染激活的分裂 - 内含肽介导的报告系统(VISI),并开发了一种直接模型,用于活细胞监测HCV细胞间传递离体:共培养 HCV感染的供体细胞(红色信号)与未感染的受体细胞(绿色信号)和通过在上清液中加入有效的中和抗体AR3A消除无细胞的传递。 利用该模型,可以通过计数受体细胞的绿色信号指定的病灶数来评估细胞间传递的效率。

【背景】越来越多的证据证明病毒可以在受感染的组织中使用不同的传播途径(Sattentau,2008; Zhong et al。,2013)。对于HCV传播,无细胞传播和细胞间传播均可介导肝细胞之间的病毒转移。虽然无细胞传播引发HCV感染,但认为细胞 - 细胞传递直接将HCV转移至相邻的肝细胞。它提供了抵抗中和抗体并有助于病毒持久性的极好方法(Brimacombe et al。,2011; Xiao et al。,2014)。之前的文章也证明了一些促进细胞传递的宿主因子,如清道夫受体BI(SR-BI),CD81,紧密连接蛋白claudin-1(CLDN1),Occludin(OCLN),表皮生长因子受体(EGFR)。 (Witteveldt et al。,2009; ...

Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation
Author:
Date:
2018-07-05
[Abstract]  Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. We developed a fixation protocol for cultured cells that preserves cytonemes, which allows for immunofluorescent analysis of endogenous and over-expressed proteins localizing to the delicate cellular structures. [摘要]  培养细胞的免疫细胞化学是用于确定细胞结构内蛋白质的组成和定位的常用且有效的技术。 然而,传统的培养细胞固定和染色方案不能有效地保存培养的细胞色素,长期专门用于形态发生转运的丝状伪足。 结果,进行了有限的机械审讯以评估其监管。 我们开发了一种用于培养细胞的固定方案,该方案保留了细胞质,允许对内源性和过表达的蛋白质进行免疫荧光分析,这些蛋白质定位于脆弱的细胞结构。

【背景】Cytonemes被分类为薄的(~200nm直径)基于肌动蛋白的丝状伪足,长度超过2μm,可以转运形态发生素(Ramírez-Weber和Kornberg,1999)。这些信号结构首先在发育中的 Drosophila 翼成像盘中进行了详细分类和描述,随后在小鼠,小鸡和斑马鱼模型生物中进行了观察(Ramírez-Weber和Kornberg,1999; Sanders et al。,2013; Stanganello et al。,2015)。在大多数情况下,只有对过表达的荧光标记蛋白进行实时成像才能进行细胞色素检测。由于传统的固定方案未能保存这些脆弱的细丝,因此对培养细胞的细胞色素的检查受到限制。这些并发症一直是决定在发育和组织稳态期间驱动细胞色素形成和功能的细胞机制以及确定这些过程是否在疾病中被破坏的限制因素。

为了克服这些限制,我们开发了一种基于修饰电子显微镜固定剂(MEM-fix)的方案,该方案可以保留培养细胞的细胞质。 ...

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