Name: quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts
Uploaded: 2012-08-27T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

这项工作是从西弗吉尼亚大学玛丽巴布伦道夫癌症中心捐赠基金的支持。我们感谢“穆勒Susette（乔治城大学）和劳拉·凯利早期的意见和协助。西弗吉尼亚大学的显微成像设备的使用（支持玛丽巴布兰多夫癌症中心，国立卫生研究院资助P20 RR16440，P30 RR032138和P30 GM103488）表​​示感谢。

1。俄勒冈州绿色生产488-明胶涂层盖玻片<ol><li>准备一个未标记的5％（w / w的）库存明胶/蔗糖溶液加入1.25克明胶和1.25克蔗糖在PBS中至终体积为50ml。的股票明胶溶液预热至37°C，并确保它完全融化之前使用。最终混合物在4℃下存放</li><li>清洁直径为13mm的第1玻璃盖玻片放置到24孔塑料组织培养板的各孔中的一个单独的盖玻片。向每孔中加入500μl的20％硝酸中，并温育30分钟。吸液的硝酸溶液，并用...

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辅助能力，可视化细胞降解细胞外基质中发现的早期步骤侵袭细胞的分子机制。陈文添在20世纪80年代初4,14,15首创，涂料荧光标记的细胞外蛋白质随后的微观分析的盖玻片上已经出现的主要技术评估侵袭伪足的功能范围广泛的细胞类型。规定的协议演示的基本的方法，用于制备明胶包被的盖玻片，形成一个小于2微米厚的胶原层适合于检测到的细胞外基质降解由细胞中最常规的荧光和激光?...

<table border="1"><tbody><tr><td> 试剂/仪器名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>明胶</td><td>西格玛</td><td> G1890 </td><td>猪皮肤</td></tr><tr><td>蔗糖</td><td>费舍尔</td><td> BP220 </td><td></td></tr><tr><td> 12毫米盖玻片</td><td>费舍尔</td><td> 50-121-5159 </td><td></td></tr><tr><td> 24孔培养板</td><td>费舍尔</td><td> 08-772-1 </td><td> BD猎鹰</td></tr><tr><td>硝酸</td><td> Ricca </td><td> R5326000 </td><td> 20％的溶液</td></tr><tr><td>聚-L-赖氨酸</td><td>电子显微镜科学</td><td> 19320-B </td><td> 0.1％的溶液</td></tr><tr><td>戊二醛</td><td>西格玛</td><td> G7526 </td><td> 8％的溶液</td></tr><tr><td俄勒冈绿488-共轭明胶</td><td> Invitrogen公司</td><td> G-13186 </td><td></td></tr><tr><td>硼氢化钠</td><td>西格玛</td><td> 213462 </td><td></td></tr><tr><td>的Triton X-100的</td><td>费舍尔</td><td> BP151 </td><td></td></tr><tr><td>罗丹明 - 鬼笔环肽</td><td> Invitrogen公司</td><td> R415 </td><td></td></tr><tr><td>反皮层蛋白（克隆4F11） </td><td>密理博</td><td> 05-180 </td><td></td></tr><tr><td> ANTI-FLAG抗体</td><td>密理博</td><td> MAB3118 </td><td></td></tr><tr><td>的Alexa Fluor 647羊抗鼠IgG </td><td> Invitrogen公司</td><td> A21235 </td><td></td></tr><tr><td>延长Gold抗淬灭</td><td> Invitrogen公司</td><td> P36930 </td><td></td></tr><tr><td> LSM 510共聚焦显微镜</td><td>蔡司</td><td></td><td></td></tr><tr><td> ImageJ软件</td><td>公共领域</td><td></td><td> <a href="http://www.macbiophotonics.ca/imagej/" target="_blank">http://www.macbiophotonics.ca/imagej/</a> </td></tr><tr><td> AutoQuant X2.2软件</td><td>媒体控制论</td><td></td><td></td></tr><tr><td> NIS Elements软件</td><td>尼康</td><td></td><td></td></tr></tbody></table>

quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts

局部组织的细胞浸润到是一个重要的发展和平衡的过程。 malregulated入侵并随后细胞运动是多个病理过程，包括炎症，心血管疾病和肿瘤细胞转移1的特征。 focalized蛋白水解降解的胞外基质（ECM）的组件中的上皮细胞或内皮基底膜是启动细胞侵袭的一个关键步骤。在肿瘤细胞中，已确定， 在体外分析广泛ECM的降解是通过腹侧肌动蛋白富集膜凸结构称为侵袭伪足2,3。侵袭伪足形式在紧密合到的ECM，在那里它们通过的作用，基质金属蛋白酶（MMPs）中度ECM击穿。肿瘤细胞的能力，以形成侵袭伪足直接相关与标注侵入本地基质和相关的血管成分3。 _content“&gt;可视化的侵袭伪足介导的细胞外基质降解的细胞荧光显微镜用染料标记的基质蛋白涂在玻片上已经成为最流行 ​​的技术评估基质蛋白水解的程度和细胞侵袭能力4,5。在这里，我们描述一个版本产生荧光标记的玻片上，利用商业可用的俄勒冈绿-488明胶共轭的标准方法，这种方法很容易扩展到迅速产生大量的涂盖玻片，我们中经常遇到的一些常见的微观文物在此过程中，如何将这些可避免的。最后，我们使用现成的电脑软件，允许标记的明胶基质的降解介导的单个细胞和整个细胞群的量化描述的标准化方法。所描述的程序提供了准确和可重复的监测能力侵袭伪足活性，也可以作为一个平台，用于评估调制蛋白的表达或测试抗侵入性的胞外基质降解的化合物对单和多细胞设置的疗效。

Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

我们描述了典型的方法涂有荧光可视化侵袭伪足介导的基质降解明胶的生产显微镜的盖玻片。利用现有的软件的计算技术，提出了一个混合的人口和量化结果由单个细胞内的基质蛋白水解为多细胞群体，涵盖整个微观领域。

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Research

JoVE Journal

Biology

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

科研

生物学

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

基于探针实时荧光定量PCR途径的小分子RNA定量检测

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

细胞外离子流的使用离子选择性自引用的微电极技术测量

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

高效的哺乳动物细胞表达和细胞外糖蛋白的结晶一步纯化

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...