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Gelatin from cold water fish skin

Company: Sigma-Aldrich
Catalog#: G7765
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Isolation of Microvascular Endothelial Cells
Author:
Date:
2018-06-20
[Abstract]  The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. Murine endothelial cell culture is an important tool for cardiovascular disease research. This protocol demonstrates a quick, efficient method for the isolation of microvascular endothelial cells from murine tissues without any special equipment. To isolate endothelial cells, the lung or heart were mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained was then incubated with an anti-CD31, anti-CD105 antibody and with biotinylated isolectin B-4. The endothelial cells were harvested using magnetic bead separation with rat anti-mouse Ig- and streptavidin-conjugated microbeads. Endothelial cells were ... [摘要]  血管内皮是正常血管稳态所必需的。其功能障碍参与各种心血管疾病。小鼠内皮细胞培养是心血管疾病研究的重要工具。该协议演示了一种快速,有效的方法从小鼠组织中分离微血管内皮细胞而无需任何特殊设备。为了分离内皮细胞,将肺或心脏机械切碎并用胶原酶和胰蛋白酶进行酶促消化。然后将获得的单细胞悬浮液与抗CD31,抗CD105抗体和生物素化的异凝集素B-4温育。使用大鼠抗小鼠Ig-和链霉亲和素缀合的微珠,使用磁珠分离收获内皮细胞。扩张内皮细胞并收集用于随后的分析。这些培养物的形态和表型特征在培养10代以上保持稳定。在任何阶段没有过度生长的非内皮来源的污染细胞。

【背景】微血管内皮细胞通过调节白细胞再循环和作为T淋巴细胞的抗原呈递细胞而在免疫应答的发展中起中心作用。内皮的良好状态对血管稳态很重要。功能失调的内皮参与各种心血管疾病,包括动脉粥样硬化,血管炎和缺血/再灌注损伤(Cid et al。,2004; Wang等人,2007)。因此,体外培养的内皮细胞培养物是研究血管生理学和疾病病理学的重要工具。然而,分离原代鼠类内皮细胞被认为是特别困难的,因为大多数描述的方案涉及用消化酶灌注器官或大血管和耗时的纯化过程(Gumkowski等人,1987) 。

该协议的目的是提供一种简单的方法,不需要使用任何特殊设备即可从肺/心脏中分离和扩展内皮细胞。使用这种方法,我们以前补充了体内研究,证明了CD31信号传导在内皮细胞细胞保护中的重要性(Cheung等人,2015年)。 ...

Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
Author:
Date:
2018-02-20
[Abstract]  Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we ... [摘要]  结核分枝杆菌(Mtb)已经发展为从其宿主吸收脂肪酸。然而,直到最近,还没有可靠的方法来量化宿主细胞感染期间细菌对脂肪酸的摄取。在这里,我们描述了一种新的方法来量化细胞内杆菌对脂肪酸的摄取。我们用Mtb组成性表达mCherry感染巨噬细胞,然后用Bodipy-palmitate代谢标记它们。标记程序后,我们在蔗糖垫上分离含有Mtb的吞噬体,并用去污剂破坏吞噬体。大量洗涤后,通过流式细胞术分析分离的细菌以确定与细菌相关的Bodipy-棕榈酸酯信号的水平。使用液体培养物中脂肪酸摄取缺陷的Mtb突变株,我们确定该突变体在巨噬细胞感染期间同化比野生型菌株少10倍的Bodipy-棕榈酸酯。脂肪酸摄取的这种定量方法可用于进一步鉴定参与细胞内Mtb和可能的其他细菌的脂质摄取的途径。

【背景】结核分枝杆菌(Mtb)同化宿主来源的脂质(脂肪酸和胆固醇)的能力使得病原体能够在其宿主内存活(Russell等人,2010; Lovewell 等人,2016)。在小鼠感染期间和在人肺组织中,通过巨噬细胞内的Mtb上调胆固醇和脂肪酸代谢相关基因来支持该想法(Schnappinger等人,2003; Rachman等人,2006; Rohde等人,2007;Fontán等人,2008; Tailleux等人,2008; Homolka et al。,2010; Rohde et ...

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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