This work was funded by the German Ministry of Defense (Grant No. M-SAB1-6-A009).

以下协议描述为早期内皮细胞迁移的调查技术。合适的培养的血管内皮细胞的需要的细胞培养瓶中用明胶预涂布，以确保内皮表型的适当扩散和维护。 细胞培养烧瓶的1.预涂层<ol><li>溶解明胶的0.1M PBS中的0.1％的最终浓度。 </li><li>高压釜中的液体灭菌参数解决方案（详见具体的高压釜的说明）。 </li><li>添加蒸压明胶溶液具有足够的体积，以无菌细胞培养瓶（ 例如，至少5毫升一T25细胞培养烧瓶）中。 </li><li>转移烧瓶成培养箱中（37℃，不需要的5％的CO 2，但不干扰）至少30分钟。 </li><li>除去剩余的明胶溶液。使用预涂层烧瓶随后（参见细胞培养）或s无菌条件，直至使用下撕毁。 </li></ol>早期内皮细胞2.细胞培养注:来自胚胎干细胞的早期内皮细胞（EEC）从分化的鼠胚体如前面10,11描述得到由PECAM-1阳性细胞级分的磁性活化细胞分选。 <ol><li>培养EEC在明胶包被的细胞培养皿中的DMEM补充有15％FCS，50U / ml青霉素，50U / ml链霉素，200μM的L-谷氨酰胺，100μM的β-巯基乙醇和1％非必需氨基酸。处理在无菌条件下的细胞。培养的细胞，用5％的CO 2在37℃和95％的湿度，直至亚融合（最大80％）。 </li><li>在亚融合，分裂细胞以1:5的比例。注意:内皮细胞的脱离是关键的一步。 <ol><li>收获细胞，用RT的Accutase。取出介质，冲洗用PBS和每25厘米加入1 ml的Accutase的<sup&gt; 2。 </li><li>孵育烧瓶在RT 2-10分钟，直到细胞脱离。分散细胞，并将它们传送到所期望的应用。注意:因为它发生时，接种的细胞在37℃下保存在培养箱不需要的Accutase的化学中和。但是，的Accutase活性也可以通过添加的DMEM含有FCS的降低。 </li></ol></li></ol> 3. Boyden小室注意:Boyden小室测定法是通过使用光的不透明聚对苯二甲酸乙酯刀片系统具有8微米的孔径进行。 <ol><li>预涂层的过滤器插入件（即适合细胞培养孔因此形成了boyden小室的内部）通过添加500μl的0.1％明胶的溶解在0.1M PBS中至少30分钟。 </li><li>如果细胞暴露于有毒化学品，根据具体的实验设计之前收获细胞暴露。注意:将细胞暴露于12.5微克丁酸氮芥/ ml的DMEM中24小时。关于具体的实验设计，指令可能会发生变化。 </li><li>收获EEC和通过使用计数池室确定的细胞数。注意:自动计数装置都可以使用，但应谨慎使用:手动细胞计数是更准确，强烈建议。 </li><li>加入500微升的细胞培养基进入Boyden小室的下腔室隔室。 </li><li>加准确的10 4欧洲经济共同体在每滤芯500微升的细胞培养基中的上腔室中。消除泡沫。 </li><li>孵化在孵化器的滤芯整整8小时。 </li><li>用PBS冲洗一次，并更换用0.5ml 4％多聚甲醛的介质在两个室25分钟，进行细胞固定。广泛冲洗过滤器，但至少3次用0.1M PBS中。 </li><li>消费用手术刀膜。 </li><li>将两片玻璃盖玻片之间的膜含有DAPI核stainin安装介质G。注意方向。确保只有已迁移朝向膜的下室侧的细胞进行计数。 </li><li>算已迁移朝向膜的下室侧与在放大400倍荧光显微镜的细胞。不要混淆膜孔与迁移细胞（ 图1A，1B）。研究生物复制合理数量（每状态至少3个生物学重复）。 </li></ol> 4.伤口愈合实验<ol><li>根据可用的设备，精心选择了细胞培养皿或板:当使用DIC的显微镜，避免塑料表面基于培养皿或孔板而是用玻璃基础上的，而不是设备。 注意:如果使用相差显微镜，是根据塑料盘也可使用。 </li><li>在合适的细胞培养装置，培养欧洲经济共同体（ 例如，4厘米玻璃底培养皿适于活细胞成像），直到80％汇合。重要提示:不要培养细胞来完成汇合。 </li><li>从头无菌10微升枪头的单层。轻轻推动尖端没有太大的压力到所述餐具表面和用0.1M PBS中移动它在一条直线上平稳地从一侧到other.Wash细胞两次以除去分离的细胞。 </li><li>添加介质的足够体积到培养皿。如果适用，添加要追究化合物。 注意:含有1.5毫升培养基15纳克/毫升的α-亚麻酸的溶液。 </li><li>安装能够活细胞成像显微镜下培养皿。确保5％CO 2，37℃和潮湿气氛。 注意:加湿是特别重要的，以避免介质的蒸发。 </li><li>超过24小时采集时间推移图像以10分钟的间隔。规划大文件大小。注:512×512像素的分辨率通常是足够的;然而，我们建议使用至少1024×1,0图片24。 </li><li>使用该软件的设置有显微镜的长度工具测量间隙宽度在t = 0小时和在t = 24小时，或者使用开放源码软件（ 例如，ImageJ的）。注意:通常特定图像采集和分析软件是由制造商提供。因此，对于使用该软件的技术细节，请参考手册。 </li></ol> 5.细胞跟踪<ol><li>根据可用的设备，精心选择了细胞培养皿或板:当使用DIC的显微镜，避免塑料表面基于培养皿或孔板而是用玻璃基础上的，而不是设备。 注意:如果使用相差显微镜，是根据塑料盘也可使用。 </li><li>种子5×10 4欧洲经济共同体在补充的DMEM在合适的细胞培养装置（ 例如，24多孔板），并培养所述细胞，用5％的CO 2在37℃和95％湿度下1-2天。 </li><li>当细胞已经长大到80％汇合，除去介质和培养细胞与新媒体的相应测试物质的存在（例如，12.5微克苯丁酸氮芥/ ml的DMEM）。始终包括对照细胞（用溶剂， 例如，乙醇处理）在37℃下在一段时间内的时间（24小时，这取决于各个测定系统上）。 </li><li>安装能够活细胞成像（37℃，5％CO 2和95％湿度），在显微镜下的培养皿。收购时间推移图像超过24小时，在预定的时间间隔。获得以10分钟的间隔图像。 </li><li>执行手动跟踪EEC中所使用的插件的ImageJ的MTrackJ。从视场中选择10个细胞，随机，通过加入每点一个数据点在时间上使用"添加"MTrackJ的命令跟踪他们的运动。 </li></ol>注:MTrackJ是免费提供的ImageJ的和是availab [Meijering， Mtrackj"http://www.imagescience.org/meijering/software/mtrackj/"。]在乐[Rasband， ImageJ的。"http://rsbweb.nih.gov/ij/"。关于MTrackJ插件的详细手册可在"http://www.imagescience.org"。 6.活细胞成像/线粒体膜电位评估<ol><li>在合适的细胞培养装置，培养欧洲经济共同体（ 例如，4厘米玻璃底培养皿适于活细胞成像）到80％汇合。 重要提示:不要培养细胞来完成汇合。 </li><li>如果适用，揭露细胞的化学物质。注意:将细胞暴露于12.5微克/毫升苯丁酸氮芥24小时。关于具体的实验设计，指令可能会发生变化。 </li><li>制备四甲（TMRM）在DMSO中的10mM储备溶液。避光。注意:原液可以储存在-20℃。 </li><li>稀释原液在细胞培养基中，以具有10微米的浓度的工作溶液（1:1,000稀释度）。避光并使用尽快。注意:工作溶液可保持在室温下一段时间（&gt; 1小时），但是，制备的新鲜工作溶液是强烈建议。 </li><li>加2μl的工作溶液到1ml新鲜的细胞培养基中（装载溶液）。 </li><li>通过与加载溶液更换细胞培养基加载细胞。孵育15分钟，在37℃，5％CO 2的加湿气氛（孵化）。注意:几乎所有的荧光指标是由活细胞随着时间的推移出口;因此，避免长时间加载或延迟分析。 </li><li>无需水洗，将适用于活细胞成像显微镜下的菜。重要提示:荧光指标对光非常敏感，因此，避免不必要的曝光。 </li><li>获取图像而无需改变采集参数，以确保不同的图像之间的可比性。 </li></ol>

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The authors have nothing to disclose.

皮肤接触有毒化学品往往导致严重的伤口愈合障碍。基本的机制在很大程度上是未知。伤口愈合是一个复杂的过程，它由不同的阶段（止血，发炎，增殖和重塑）的。细胞迁移涉及每个阶段，然而，它是最重要的肉芽组织的形成。这里，新血管通过血管造影或血管生成要么形成。 这两个过程都需要前体和早期内皮细胞不受影响迁移。细胞迁移和明确的干扰细胞迁移的基本机?...

细胞迁移是许多生理和病理过程，包括发展，各种疾病及皮肤受伤后伤口愈合很重要的。 以下皮肤损伤，炎症消除受损或坏死组织及肉芽驱动器初步伤口缝合，并允许通过血管形成新的毛细血管网络的（小说形成）或血管生成（已有的囊泡萌芽）1-3。既vasculo-和血管生成需要内皮细胞的迁移。越来越多的血管网络是必不可少的运输氧和营养物质的角质形成细胞增殖，最终发生角化，形成新的上皮细胞，并提供伤口缝合。 内皮细胞受损迁移是伤口愈合紊乱4,5的一个根本原因。早期的内皮细胞。因此，方法来评估迁移需要探索病理生理的细胞迁移紊乱术，并确定新的策略的治疗干预。 皮肤暴露于烷化剂（ 例如，硫和氮芥子气）导致伤口愈合紊乱6。这种化合物被用作化学战剂的一些冲突，在20世纪，并保持之所以强烈关注，由于在政治上不稳定的地区和相对简单的合成现有库存。虽然硫芥在1822年首次合成，SM暴露的分子和临床病理学不被了解的细节，也没有解药SM曝光已经确定。 几项研究已经进行了解和伤口愈合不良SM曝光后模拟和测试能够保留该效果的潜在候选化合物。 Schmidt 等人（2009）测试了苯丁酸氮芥的效果，与在谋类似的SM性质的烷基化化合物本身胚体模型，发现了戏剧性，有时在血管形成7减少99％以上。这种不利影响是最有发展的哪个，在生理条件下，通过增殖和血管内皮前体细胞的迁移支配阶段明显。因此，这些细胞被鉴定为特别敏感烷化剂。 Steinritz 等人（2010）测试了活性氧簇（ROS），特别是N-乙酰半胱氨酸（NAC）和阿尔法亚麻酸（ALA）清除剂其减少的SM毒性在小鼠胚体模型和具体地，涉及能力恢复血管形成8。观察临时保护作用，表明过度ROS形成是可能有助于SM的对伤口愈合的不利影响。这些影响是不是永久性的，这两个候选化合物可能不能够恢复在长期8血管形成和伤口愈合的。 HoweveR，这些实验在一个复杂的三维模型，确实让细胞迁移的调查进行的。因此，我们随后被测试的NAC和ALA对于具有在血管形成9的过程中起关键作用的欧洲经济共同体的细胞迁移的有益效果。 此外，有证据表明，细胞极性所需的细胞迁移。线粒体功能障碍导致ROS形成被证明损害细胞极性和可因此细胞迁移有负面影响。因此，活细胞成像对于线粒体功能被执行，并且进行了检查的ROS清除剂的作用。以下协议描述为EEC，Boyden小室法，伤口愈合实验，包括细胞跟踪分析和使用TMRM的详细线粒体功能评估的培养一般要求。对于EEC种植和移植实验方案的重要方面突出。

<table><tbody><tr><td>Boyden Chamber&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 3 &amp;micro;m</td><td>Corning Incorporated</td><td>#351151</td><td></td></tr><tr><td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 8 &amp;micro;m</td><td>Life Sciences</td><td>#351152</td><td></td></tr><tr><td>(for use with Falcon Insert 24 well Companion Plate (353504)</td><td></td><td></td><td></td></tr><tr><td>Wound&amp;nbsp; healing assay</td><td></td><td></td><td></td></tr><tr><td>Glass bottom dishes</td><td>Word Precision Instruments, Inc.</td><td>#FD35-100</td><td></td></tr><tr><td>Assessment of mitochondrial potential</td><td></td><td></td><td></td></tr><tr><td>TMRM (tetramethylrhodamine methyl ester)</td><td>Life Technologies</td><td>#T669</td><td></td></tr><tr><td>Cell culture</td><td></td><td></td><td></td></tr><tr><td>Accutase</td><td>PAA, Pasching, Austria</td><td># L11-007</td><td></td></tr><tr><td>&amp;alpha;-Linolenic acid</td><td>Fluka (Sigma), Steinheim, Germany</td><td>&amp;nbsp;# L2376</td><td></td></tr><tr><td>Chlorambucil</td><td>Fluka (Sigma), Steinheim, Germany</td><td># 23125</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich, Steinheim, Germany</td><td># G2500-100G</td><td></td></tr></tbody></table>

assessment of endothelial cell migration after exposure to toxic chemicals

接触化学物质（包括如硫和氮芥子气烷化剂化学战剂）会导致过多的临床症状，包括伤口愈合障碍。伤口愈合的生理过程是非常复杂的。肉芽组织的形成是在这个过程中产生了初步伤口闭合和提供的新的毛细血管的网络中的关键步骤 - 无论是通过血管（新形成）或血管生成（出芽现有船只）。无论vasculo-和血管生成需要内皮细胞的功能，定向迁移。因此，早期内皮细胞（EEC）迁移的调查是重要的，了解化学诱导伤口愈合障碍的病理生理学和潜在确定新的战略治疗干预。 我们评估伤口愈合不良烷化剂暴露后测试潜在的候选化合物TReatment。我们使用了一组在此协议中概述的技术。一个改良的Boyden室定量调查EEC的化学激活描述。此外，使用伤口愈合实验，结合轨道分析，以定性评估迁移的说明。最后，我们证明了使用的荧光染料TMRM对于线粒体膜电位的调查，以确定潜在的干扰细胞迁移的机制。下面介绍的协议已被改编为EEC的调查基本技术。

皮肤暴露于烷化剂引发红斑，水疱形成和与伤口愈合病症相关皮肤溃疡。伤口愈合需要其基于内皮细胞迁移的血管造影和血管。定量迁移可以通过使用Boyden小室测定法进行评估。如图欧洲经济共同体的图1C暴露于烷化剂氮芥导致细胞迁移9一显著降低。除了接触苯丁酸氮芥24小时后直接活性氧清除剂的α-亚麻酸（ALA）的显著救出这种表型，几乎控制水平。 ?...

Watch this Scientific Journal Video about Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals at JoVE.com

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

assessment-endothelial-cell-migration-after-exposure-to-toxic

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.6K 次观看.  Bundeswehr Institute of Pharmacology and Toxicology. Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

视频: Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

细胞侵袭和迁移过程的评价: 视频 Microscope-based 刮伤法与博伊登室法的比较

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

科研

癌症研究

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

生物工程

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

人中性粒细胞迁移的跨越培养膀胱上皮定量评价

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

免疫与感染

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

人类干细胞衍生内皮细胞和GABAergic神经元的迁移、化学吸引和共培养测定

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

神经科学

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

生物学

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

经济高效且适应性强的划痕迁移检测

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

An Introduction to Cell Motility and Migration

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues and organs during development, but on the other hand, the same mechanisms are used by tumor cells to move and spread in a process known as cancer metastasis. One of the primary cellular machineries that make cell movement possible is an intracellular network of myosin and actin molecules, together known as &#8220;actomyosin&#8221;, which creates a contractile force to pull a cell in different directions.In this video, JoVE presents a historical overview of the field of cell migration, noting how early work on muscle contraction led to the discovery of the actomyosin apparatus. We then explore some of the questions researchers are still asking about cell motility, and review techniques used to study different aspects of this phenomenon. Finally, we look at how researchers are currently studying cell migration, for example, to better understand metastasis.

Cell Motility and Migration, Cell Invasion and Chemo-attraction

导论细胞运动和迁移

Cell motility and migration play important roles in both normal biology and in disease. On one hand, migration allows cells to generate complex tissues ...