Name: a simple bioassay for the evaluation of vascular endothelial growth factors
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SAS and MGA are supported by Project Grants, a Program Grant and Research Fellowships from the National Health and Medical Research Council of Australia (NHMRC), and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. MMH has support from a Peter MacCallum Foundation Grant.

IL-3和制备的源WEHI-3D​​的空调中等注意:小鼠粒细胞白血病细胞系WEHI-3D​​是培养以产生含IL-3条件培养基。 <ol><li>培养WEHI-3D​​中的Dulbecco改进的Eagle培养基（DMEM），10％胎牛血清（FBS），1％的长寿命补充谷氨酰胺，50μg/ ml的庆大霉素。接种5×10 6个细胞中生长的对数相到50ml在一个T175 cm 3的组织培养瓶的新鲜培养基并生长约7天，或者直到细胞已经由约24通过增长其数期- 48小时（避免过度培养细胞死亡）。条件培养基是能够被存储为多个年在-20℃，所以200批次 - 400毫升能在同一时间产生。 </li><li>滗析从烧瓶和旋转流体和细胞在1,000×g下15分钟以除去细胞和细胞碎片。拆下顶部90％supern的atant。通过0.22微米的过滤器单元进行过滤。 </li><li>等分试样WEHI-3D​​的条件培养基（CM）在1ml 50毫升，并在-20℃下200毫升卷进行存储或-70℃。小等分试样用于测定制备，用于制备培养基为因子依赖性细胞系的通道中间的卷，以及用于长期储存的卷是有用的。 IL-3是在4℃下，如果在无菌条件下使用，所以解冻介质可以存储几个星期相对稳定。 </li><li>另外，使用重组小鼠IL-3的稀释于培养基中，通过0.22微米的过滤器单元过滤后50ng / ml的水平。 </li></ol> 2.培养和评价的VEGFR-2-EpoR的-的Ba / F3和控制的Ba / F3细胞系的<ol><li>培养控制Ba / F3细胞在DMEM，10％FBS，50μg/ ml的庆大霉素或青霉素/链霉素补充，1％稳定的L-谷氨酰胺和10％WEHI-3D​​-CM。有关从细胞每三天传代细胞以1:15稀释在对数期生长。 </li><li>培养VEGFR2-EpoR的-Ba / F3细胞在DMEM，10％FBS，50μg/ ml的庆大霉素，1％稳定的L-谷氨酰胺和10％WEHI-3D​​-CM和1毫克/毫升G418。传代细胞1:15稀释在对数期生长的细胞。有关嵌合受体的构建和表达的更多详细信息，请参阅24。 </li><li>收获控制的Ba / F3或VEGFR2-EpoR的-Ba / F3细胞中旬对数期的文化。轻轻吸取从烧瓶底部除去这些非贴壁细胞。在小鼠张力磷酸盐缓冲盐水，pH值7.3（PBS）中洗涤三次，（10毫升，离心，在750×g下5分钟以回收细胞沉淀）以除去含IL-3培养基。 </li><li>洗涤细胞用DMEM和添加剂一次，而不WEHI-3D-CM或重组IL-3，然后重悬在该培养基中以7.4×10 4个细胞/ ml（即每13.5微升1,000个细胞的浓度; 10,000个细胞每135微升通过使用血细胞计数器计数测定）为在分别测定的半定量或定量的版本使用。 </li><li>评估由台盼蓝排斥（警告）的活力细胞。混台盼蓝的PBS（0.4％）1:1的细胞群，并在血细胞计数器计数的最低100个细胞。这样会占用染料细胞被认为是死亡或濒临死亡。的大于98％的生存力是需要进行测定。 </li></ol> 3.半定量分析<ol><li>添加包含在13.5微升IL-3缺陷型培养基中以在RT 72孔微孔板的孔中洗涤细胞（1000细胞）。小心aliquotting确保细胞沉淀在重力作用下不偏细胞浓度时混合细胞悬液。使用以及校准P20自动化移液器和蒸压提示。 </li><li>添加测试样品和对照的孔中在10％体积（1.5微升，使得每孔含有1,000个细胞15微升的最终体积）使用校准P20移液管或，优选，P2移液管。以CA重，以确保样品在pH值，盐和其他可能的细胞毒性/细胞抑制剂的物质而言的培养条件为Ba / F3细胞兼容。如果可能的话，使用兼容的介质或缓冲液（如 DMEM或PBS，分别），或在此类介质或缓冲液稀释。研磨用相同的尖端将确保混合。 </li><li>对于对照样品，包括（i）中单独的含无IL-3; （ⅱ）WEHI-3BD-CM加入到培养基单独以10％的最终体积和/或（iii）VEGF-A稀释至在单独介质100纳克/毫升，不含有IL-3。 </li><li>填用无菌水，PBS或培养基的微孔板的任何未使用的孔中，放置的潮湿容器中的板（与水浸泡棉纸）允许气体交换。孵育在容器内的细胞在10％的CO 2的潮湿气氛中。该测定可以舒适地设置在3小时由一个人如果测试样品和媒体组件是可用的。 </li><li>评估板通常后16小时在培养其阳性和阴性样品之间的时间区别是显而易见的。参见图3的细胞如何出现在文化的例子。 100×放大率 - 使用标准倒置相差显微镜在40分析平板。 注意:包含韦尔斯支持生长因子如IL-3或VEGF-A将含有呈圆形和半透明的细胞。而不支持生长因子或与单独的培养基孔中会具有降低的圆形细胞，以及死亡或正在死亡的细胞和细胞碎片（例如， 图3C）的号码。在24-48小时后孵化评估法是最佳的灵敏度。 </li></ol> 4.定量生物测定<ol><li>在IL-3缺陷型培养基中添加洗涤细胞（10,000个细胞在135微升），以在RT标准的96孔微量滴定板的孔中。小心aliquotting以确保重力不影响CE细胞沉淀过程中，混合细胞悬液LL浓度。 </li><li>添加测试样品和对照到孔在使用校准吸管（P20）10％体积（15微升）。小心确保样品与pH值，盐或其他潜在的细胞毒性/细胞物质方面的培养条​​件为Ba / F3细胞兼容。如果可能的话，使用兼容的介质或缓冲液（ 例如 ，DMEM或PBS），或在此类介质或缓冲液稀释。过滤器采用0.22微米孔径的醋酸纤维素离心管过滤装置灭菌小容量。 </li><li>孵育细胞，生长因子和/或抑制剂剂的混合物中的10％的CO 2的潮湿气氛48小时。具有水渍薄纸，并允许气体交换的潮湿容器中进行测定。在温育期结束时，评估使用的，低于该列出的替代性方法中的一种测定是在井的活细胞数目的替代标志。 </li><li> 替代＃1:3 H-thymidiNE公司注册 注意:此量化是专为96孔板形式。 <ol><li>在37℃下（每孔150μl的体积）为48小时测定板的温育后，在50微升测定培养基的体积添加3 H-胸苷（的Ba / F3培养基没有IL-3 / WEHI-3D ​​CM）每孔以20微居里/ ml的浓度，得到的每孔1微居里的最终剂量。 </li><li>孵育板用于使用细胞收集器收获细胞前在37℃下再进行4小时。通过将过滤器在闪烁体提取各个样品，并用液体闪烁计数器定量。 </li></ol></li><li> 替代＃2:细胞内ATP生物发光检测 注意:此方法使用当与细胞裂解物相结合，可产生反映在人口中的活细胞的生物发光信号的ATP的监测试剂。 <ol><li>裂解细胞以提取ATP和使用ATP监测试剂，以产生一个根据制造商的说明发光信号。使用带有一个96孔板格式兼容光度计读取发光。 </li></ol></li><li> 替代＃3:由活细胞的指示剂染料酶催化还原 注:基于刃天青试验显示良好的相关性细胞活力。 <ol><li>结合培养细胞用刃天青染料和量化使用根据制造商的说明在荧光读板器测定。 </li></ol></li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+catalogue+of+somatic+mutations+from+a+human+cancer+genome.">A comprehensive catalogue of somatic mutations from a human cancer genome.</a> Nature. 463, 191-196 (2010).</li></ol>

史蒂芬A.堆和Marc M.亚琛是股东Circadian技术有限公司，公司通过瞄准生长因子VEGF家族发展疗法。

此处所描述的测定法依赖于使用高存活率的细胞，这是依赖于生长因子。因此细胞需要仔细培养，以确保它们是因子依赖性的，并保留嵌合受体的表达。确保介质新鲜的，而不是存放过长的时间，并且WEHI-3D​​ CM为高活性是重要的。细胞需要从含培养基的IL-3彻底洗涤进测定培养基，以确保无残留的IL-3将细胞暴露于配位体抢救时污染测定。作为配体可以来以多种不同的形式，护理需要用于测定制?...

血管内皮生长因子（VEGF）家族的分泌蛋白的生长因子和它们的同源细胞表面受体是可溶性配体和膜包埋的受体，分别在穿过细胞膜转导信号该函数的一个重要和多样的基团。它们主要发挥作用的内皮细胞，而且在和上皮来源的细胞的那些免疫系统1,2。信令由配体激活的VEGF受体（VEGFRs）是在主要的病状，如年龄相关性黄斑变性和癌症关键的，和治疗啮合途径靶向它们是在常见的临床使用（例如，单克隆抗体贝伐单抗靶向的VEGF-A）的3,4。 一项所述的VEGF家族的复杂性是存在于自然界（可溶性配体VEGF-A，VEGF-B，VEGF-C，VEGF-D，由parapox病毒家族ORF和蛇毒VEGF，加上其他编码的VEGF蛋白质的多样性抑制VEGF-A）2亚型。 这些配体与受体酪氨酸激酶家族的三个成员，即VEGFR-1，VEGFR-2和VEGFR-3相互作用。这些受体可变地表达于不同的细胞类型，但该行的所有大小5的血液和淋巴管内皮细胞的表面上常常共表达。 VEGFR-2可以结合哺乳动物配体VEGF-A 6，VEGF-C 7和VEGF-D -8,9-以及口疮病毒VEGF 10和蛇毒VEGF的11。 VEGFR-2在驱动血管发生在胚胎发育（新血管从预先存在的血管的生长），伤口愈合，癌症和眼病主要作用。在这些情况下，配体例如VEGF-A，-C和-D结合并激活血液血管内皮细胞12-15受体。上淋巴管内皮细胞，VEGFR-2在淋巴管生成的作用，新的淋巴管16的形成。 VEGFR-2还可以促进扩张和主要动脉和淋巴管在健康组织和疾病17扩张。 VEGFR-2的完整的理解:配体的相互作用是因此对于抑制剂的开发用于治疗血管生成依赖性疾病18重要。而VEGF-A结合的对VEGFR-2的大部分同种型，是必需的VEGF-C和VEGF-D的蛋白水解裂解，以释放由表现出高亲和力结合VEGFR-2 19,20的VEGF-同源结构域的片段。 我们已经开发出一种生物测定来监测，其被设计来规避初级内皮细胞，这是技术上难以通过，购买昂贵的和培养的需要VEGFR-2的配体（需要专门介质）21和表达多种VEGFRs和相关的共受体22。旨在螺柱当与其他VEGF受体或共受体VEGFR-2的异源可引起不希望的复杂性ý二进制受体-配体相互作用，评估活性归属于特定的受体，或评估抑制试剂的效果。23。生物测定保留在细胞膜相关受体的流动性，并允许的配位体的结合能力并交联的VEGFR-2的细胞外区域的评价。 生物测定依赖于其中（在这种情况下，VEGFR-2）VEGF受体的胞外区融合至跨膜和促红细胞生成素受体（EpoR的），细胞因子受体家族的一个成员的胞内区域的创建的嵌合受体8,24。此融合蛋白然后在从属因子原B细胞系的Ba / F3表示，在其上的刺激与能结合和交联的受体的胞外结构域导致胞质效应区，其能够激活一个配位体通过JAK激酶（JAKS）转导存活信号，促进细胞的存活和/或增殖。与此相反，全长VEGFR-2在相同的细胞类型，并刺激配体表达，不促进细胞存活和增殖，表明VEGFR-2通路的近端信令效应不在此细胞类型可用。 我们已经使用该测定在各种情况下，探索新的VEGFR-2的配体10,19,20,24-29的结合。在与VEGFR-3-EpoR的-的Ba / F3测定结合，我们比较了VEGF-C的结合和交联的VEGFR-2和VEGFR-3 30的相对活性和VEGF-D的生长因子。该测定法已被用于表征中和单克隆抗体对VEGFR-2或VEGF-D，可溶性VEGFR-2捕集和拟肽靶向VEGF家族31的抑制活性。该试验也用于显示的VEGF的不同ORF病毒株结合交联的VEGFR-2在测试之前在初级内皮细胞的能力，并<s达&gt; 10,26。该测定是对的VEGF的突变体的快速筛选，可以它们被引入到更费力内皮细胞分析25，或当用于净化生长评估协议因子27前被快速评估活动特别有用。 我们描述了检测容易进行，并且半定量版本允许监测的生长因子，抗体或其他实验的可溶性受体结构域的生产或纯化时有时需要快速测定。使用该测定的易用性使得它与从血液或淋巴管衍生自特定的组织或器官系统主内皮细胞进行进一步和更完全的研究，一个理想的补充。

<table><tbody><tr><td>Trypan Blue</td><td>Sigma-Aldrich</td><td>T8154</td><td>0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer</td></tr><tr><td>G418 Sulphate (Geneticin)</td><td>Invivogen</td><td>ant-gn-5</td><td>Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties.</td></tr><tr><td>&lt;sup&gt;3&lt;/sup&gt;H-Thymidine</td><td>PerkinElmer</td><td>NET-027</td><td>This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation</td></tr><tr><td>Vialight Plus Kit</td><td>Lonza</td><td>LT07-221</td><td>Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent</td></tr><tr><td>Prestoblue Cell Viability Reagent</td><td>Invitrogen</td><td>A13261</td><td>Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell</td></tr><tr><td>Nunc Minitray with Nunclon Delta Surface (72 well)</td><td>Thermo Scientific</td><td>136528</td><td>Small microtitre plate</td></tr><tr><td>96 well Tissue Culture Plate</td><td>Falcon, Corning Inc.</td><td>353072</td><td></td></tr><tr><td>DMEM (1X)</td><td>Gibco</td><td>11965-92</td><td></td></tr><tr><td>GlutaMAX (100X)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>&amp;nbsp;10099-141</td><td></td></tr><tr><td>Cell Harvester</td><td>Tomtec Life Sciences</td><td></td><td>Tomtec Harvester, 96 Mach 3M Cell Harvester</td></tr><tr><td>Liquid Scintillation Counter</td><td>LKB Wallac</td><td>1205</td><td>LKB Wallac 1205 Betaplate Scintillation Counter</td></tr><tr><td>UniFilter-96 GF/B</td><td>Perkin Elmer</td><td>6005177</td><td>White 96-well Barex Microplate with GF/B filterof 1 &amp;micro;m poresize</td></tr><tr><td>Gentamicin</td><td>Gibco, Life Technologies</td><td>15750-060</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco, Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>0.22 um pore cellulose acetate centrifuge tube filter unit</td><td>Costar, Corning Inc.</td><td>8160</td><td>Centrifuge tube filters have a 0.22 &amp;micro;m pore CA membrane-containing filter unit within a 500 &amp;micro;l capacity polypropylene microcentrifuge tube.</td></tr><tr><td>Fluorescence Reader</td><td>BioTek</td><td></td><td>BioTek Synergy 4 Hybrid Microplate Reader&amp;nbsp;</td></tr></tbody></table>

a simple bioassay for the evaluation of vascular endothelial growth factors

参与血管生物学受体酪氨酸激酶及其相互作用的配体的分析常常是具有挑战性的，由于相关的受体，范围广泛的相关配体和处理专门内皮细胞的原代培养物的难度家庭的组成型表达。这里，我们描述用于检测配体的血管内皮生长因子受体-2（VEGFR-2）中，促进血管生成和淋巴管生成的信号的键传感器生物测定。编码VEGFR-2的胞外（配体结合）区的融合与跨膜和促红细胞生成素受体（EpoR的）的细胞质区域的cDNA，在因子相关性细胞系的Ba / F3表示。该细胞系生长在白细胞介素-3（IL-3）和该因子的结果的撤出在细胞的死亡存在24小时之内。在VEGFR-2 / EpoR的受体融合的表达提供了一种替代机制，促进生存和势的在能够结合和交联的融合蛋白（ 即 ，一个可以交联的VEGFR-2的胞外区）的胞外部分的配位体的存在下稳定转染的Ba / F3细胞的LLY增殖。半定量的方法，其中配位体和细胞小体积在24小时允许快速结果，并且涉及活细胞数目的替代标志物的定量方法:在测定可以通过两种方式来执行。该测定是比较容易进行，是高度响应于已知VEGFR-2的配体，可容纳的VEGFR-2的信号，如单克隆抗体的受体或配体，和可溶性配体陷阱胞抑制剂。

在这一节中，我们显示的实验展示一VEGFR-2-EpoR的-的Ba / F3生物测定（参见图1用于测定的原理）的基本特征的结果。其他已发表 ​​的研究表明在测定的替代VEGFR-2的配体，突变的VEGF分子和抑制性单克隆抗体8,10,19,24-30的更广泛的应用。 此处呈现的数据表示，其中的VEGFR-2-EpoR的-的Ba / F3生物测定来量化三种重?...

Watch this Scientific Journal Video about A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors at JoVE.com

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

我们描述了检测，定量和监测血管内皮生长因子家族的配体的成员的活性的简单的基于细胞的生物测定法。该测定法使用在因子相关性细胞系中表达的嵌合受体的配体，以提供受体结合和交联的半定量或定量的评估。

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

a-simple-bioassay-for-evaluation-vascular-endothelial-growth

Research

JoVE Journal

Biology

9.6K 次观看.  Peter MacCallum Cancer Centre. 我们描述了检测，定量和监测血管内皮生长因子家族的配体的成员的活性的简单的基于细胞的生物测定法。该测定法使用在因子相关性细胞系中表达的嵌合受体的配体，以提供受体结合和交联的半定量或定量的评估。 

视频: A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

科研

生物学

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

发育生物学

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

免疫与感染

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

医学

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

使用划痕伤口迁移和球状体发芽测定法研究血管生成

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

作者聚焦：通过检测开发中的挑战和创新研究血管生成

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

一个简单的生物测定血管内皮生长因子的评价

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监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

用迈尔斯测定法评价皮肤血管 Hyperpermeability 诱导剂的含量

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成