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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Boyden Chamber&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 3 &#181;m</td>
			<td>Corning Incorporated</td>
			<td>#351151</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning FluoroBlok Tissue Culture (TC)-treated Inserts, 24 well - 8 &#181;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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wound&#160; 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></td>
			<td></td>
			<td></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></td>
			<td></td>
			<td></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>&#945;-Linolenic acid</td>
			<td>Fluka (Sigma), Steinheim, Germany</td>
			<td>&#160;# 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

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

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

科研

发育生物学

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

心外膜生长培养测定和<em&gt;离体</em&gt;心外膜源性细胞迁移的评估

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

将毛细血管纳入内皮细胞珠发芽试验

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

微流控法评价人诱导的多能干细胞内皮细胞 (hipsc-ec) 对白细胞粘附的评价

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...