丹麦独立研究、技术和生产理事会, 赠款 #4005-00029

实验设置没有道德和法律上的限制, 并与罗斯基勒大学进行了协商。在图1和图 2中的光学摄像头的机制中可以看到实验装置的概述。
1. 准备
<ol><li>在实验开始前, 用70% 乙醇清洗工作台和所有设备, 在无菌层流工作台执行所有步骤。</li>
	<li>将光学相机放在传统的孵化器中, 37 摄氏度, 5% CO2 , 以确保细胞的最佳条件。</li>
	<li>在 DMEM 培养基中生长 HaCaT 细胞, 其中有10% 只血清和1x 抗生素 (青霉素和链霉素) 在 T75 瓶中。</li>
	<li>使用1毫升1x 胰蛋白酶分离细胞和分培养他们每3-4 天, 通过分散它在细胞层。在使用前先加热到室温。</li>
</ol>2. 在48井微量滴定板中生成一层 HaCaT 细胞
<ol><li>用巴斯德吸管从 T75 瓶中取出培养基。</li>
	<li>在 T75 瓶中加入5毫升1x 无菌 PBS, 洗涤烧瓶中的黏附细胞。 注:确保 PBS 没有钙和镁。</li>
	<li>分发 PBS 并用巴斯德吸管将其取出。</li>
	<li>重复步骤 2.2-2.3。</li>
	<li>添加1毫升1x 胰蛋白酶到烧瓶, 并分散在细胞层。</li>
	<li>在传统的孵化器中孵化出37摄氏度的烧瓶, 直到细胞分离 (5-8 分钟足够用于大多数细胞系)。</li>
	<li>在显微镜下查看细胞是否分离。一旦细胞分离, 添加9毫升的常规培养基和10% 的血清, 以使1x 胰蛋白酶失效。</li>
	<li>用 hemocytometer、库尔特计数器或类似的计数单元格。 注:台盼蓝可用于查看死细胞。然而, 在1x 胰蛋白酶 HaCaT 细胞是完全圆的, 当活着。</li>
	<li>转移 7.5 x 104细胞/井在250µL 10% 血清介质到一个圆底的48井组织培养板材 (标准板材)。 注: 还可以在6、12、24或96井板中设置化验, 所有这些都可以在光学显微镜下测定。</li>
	<li>轻轻地将盘子从一侧移到一边, 以确保细胞在井中均匀传播, 否则细胞将聚集在水井的中心。在光显微镜下检查以确保电池在介质中均匀传播。</li>
	<li>把盘子放在传统的 CO2孵化器中, 一夜之间37摄氏度, 或者直到细胞粘在井底。</li>
</ol>3. 增加对移徙影响的刺激
<ol><li>检查油井是否在光镜下90-95% 汇合。</li>
	<li>在适量的培养基中制备10µg/毫升丝裂霉素 C。 注:在本研究中, 丝裂霉素 C 在1毫克/毫升的库存溶液中保持在4摄氏度。丝裂霉素 C 在溶液中可以储存至多一个星期在黑暗中2-8 °c。</li>
	<li>用巴斯德吸管从每个井中取出所用的介质。</li>
	<li>添加250µL 5 µg/毫升丝裂霉素 C 的每一个井。</li>
	<li>在37摄氏度和 5% CO2上孵育2小时的板材。</li>
	<li>通过稀释适量的培养基来制备肽的刺激物。 注:在这项研究中, 人和牛乳铁蛋白和乳铁素的测试在25µg/毫升的数量250µL</li>
	<li>用德霉素 C 去除培养基, 用巴斯德吸管取出。</li>
	<li>用250µL 1x PBS (室温) 冲洗井 1x, 用巴斯德吸管去除洗涤液。</li>
	<li>添加250µL 1x PBS 和手动划伤井垂直与200µL 吸管提示。为每个井使用新的吸管尖端。</li>
	<li>取出 PBS 并添加250µL 肽刺激。</li>
	<li>通过调整板两侧的螺钉, 将板安装在光学摄像系统中。把盖子放在微量滴定板上。 注:如有必要, 相机可以在盖子打开的情况下操作。</li>
</ol>4. 为计算机上的光学摄像机设置程序
<ol><li>单击左侧任务栏中的 "获取"。</li>
	<li>选择左侧任务栏中的功能伤口愈合。</li>
	<li>通过点击板块插图, 选择板块类型。</li>
	<li>取消选择未使用的井, 标记它们并单击屏幕左上角的 "启用"。 注:默认情况下, 所有井都被选中。</li>
	<li>用右键单击其中一个井, 调整井上透镜的焦距, 以适应细胞的位置。 注:自动对焦也可以手动调整, 通过移动栏的右侧的板块插图。</li>
	<li>设置图片间隔(如(00:30:00)) 中图像的时间段。</li>
	<li>将重复次数设置为适合所需周期 (例如97 重复)。</li>
	<li>通过单击右下角的 "开始" 按钮开始运行。</li>
</ol>

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作者没有什么可透露的。

移徙在发展、创伤愈合、免疫学和癌症研究中的重要性导致了研究迁移的几种不同方法。迁移可以是由单元格扩展或关闭间隙的表达式。最常见的抓痕的闭合是测试, 因为它是更容易种植细胞在单层, 然后做一个擦伤比生长的细胞在特定的形式8。我们的研究表明, 手工抓伤的优化方法, 因为该技术是易于掌握和低成本。我们介绍了 DNA 合成抑制剂丝裂霉素 C, 一个额外的清洗, 以优?...

迁移是影响几个生理方面的重要过程。在伤口愈合中, 迁移有利于皮肤的重新上皮化。在非愈合伤口, 如慢性伤口, 机会性细菌引起的伤口感染, 开放性伤口是理想的细菌生长和形成生物膜1,2。生物膜是耐药性细菌发展的主要原因之一, 是我国现代社会面临的最大威胁之一3。在伤口愈合过程中, 免疫细胞无法穿透生物膜。在癌症中, 癌细胞的迁移是转移的关键步骤, 这是4、5实体肿瘤患者死亡的主要原因。因此, 能够研究迁移是至关重要的。
擦伤检测通常用于测试细胞迁移, 因为它便宜, 易于执行黏附细胞线, 如成纤维细胞, 内皮, 和上皮细胞线6,7。今天, 有几个方法来执行划痕8, 并报告了不同的材料, 包括牙签9, 电池铲运机10, 激光11, 电流12。所有的方法都有不同的优缺点, 应该根据单元类型、预算和分析工具来确定方法。举例来说, 如果使用的细胞类型需要涂层, 手动减压,如牙签或吸管提示, 将影响在该地区的迁移, 应使用不同的方法。在本研究中, 我们将专注于手工刮擦。
手工刮擦的缺点是大小、划痕的形状和划痕边缘的单元格堆积的变化。许多协议存在, 并且一张高度被引述的纸劝告增加的速度和/或者彻底洗涤在划伤13以后。此外, 还使用了几种方法来最大限度地减少扩散, 因为细胞的增殖不能与移徙区分开来, 因此可能给移徙带来假阳性结果。一些报告的方法是血清饥饿前的14和减少的数量的血清15。然而, 这两种方法都不能确保没有扩散。因此, 我们报告一个前处理细胞丝裂霉素 C, 以确保真正的迁移结果。丝裂霉素 C 是一种抗生素, 它通过与 dna 形成共价键来抑制 dna 合成, 从而防止 dna 分离16。通过对丝裂霉素 C 的预处理, 在刮伤前用 PBS 洗涤, 检测到的间隙闭合显示了细胞的真正迁移 (图 1)。
所有方法的一个特点是需要一个系统来分析迁移17的过程。一个常见的和廉价的方法来分析的进展是使用光场显微镜拍摄的图像的划痕在预先确定的时间点, 然后分析关闭的差距在图像软件18,19。这种方法是费时的, 不可靠的关于拍照在同一地点和焦点, 和运动从孵化器到相机的力量周期的温度变化, 而拍摄。还开发了其他方法, 通过测量电流流量来检测偏移量。目前的变化, 因为细胞覆盖更多的空间在板块上, 这是读取作为增加阻抗20。通过测量阻抗, 细胞的环境不受影响, 因此该方法被认为是无侵入性的。这种方法依赖于专门的板, 金电极, 这是昂贵的, 但它们能够检测到许多过程, 如细胞黏附, 增殖, 迁移和细胞死亡。这种方法的一个很大的缺点是阻抗测量不能直接表示迁移, 因为温度等因素对阻抗有很大的影响。阻抗的变化可能是由软件没有审查的几个因素引起的, 使得对数据的分析更加困难。因此, 缺乏可视化需要常规的光显微镜来验证迁移。
为了克服现有技术的许多缺点, 我们使用实时自动光学相机。光学摄像头是一种检测系统, 具有高吞吐量的显微镜, 在一个小的平台上有透镜, 照明单元, 和数码相机。它有一个内置的软件, 它可以测试迁移和扩散等其他事情。为了检测迁移, 分别用两种算法对细胞的生长和迁移动力学进行了分析, 称为增殖和创面愈合分析。扩散分析是基于两步图像分割算法, 应用纹理分析方法, 通过高级直方图分析来检测细胞覆盖区域 (黄色) 和空背景区域之间的差异。伤口愈合分析是基于三步算法检测细胞单层, 定位伤口间隙, 确定间隙宽度。这些步骤是在用户确定的时间点进行的, 数据显示为覆盖区域、间隙宽度和间隙区域。这台机器的最大优点之一是, 它能够通过液体, 因为相机21的角度来成像粘附细胞。相机 lenstakes 倾斜的图像, 投影到 z 栈的单 z 平面生成, 以确保图片的焦点 (图 2)。z 栈是通过合并的一系列图片 (如40) 垂直于伤口边缘, 并贯穿整个伤口生成。z 栈能够捕捉到细胞层的深度, 以及贯穿伤口的聚焦平面。此外, 该系列图像可以放在一起的图像视频收集, 点击一个按钮。
我们演示了一个优化的协议, 以检测角质形成细胞线 HaCaT 的迁移。我们测试了乳铁蛋白及其 n-端肽, 乳铁素, 从人类和奶牛, 分析其对迁移的影响, 因为这些分子已经证明有许多应用作为抗菌和免疫调节分子22,23. 将四种化合物与未经处理的 HaCaT 细胞进行比较, 并将表皮生长因子 (EGF) 用作阳性对照。

<table><tbody><tr><td>oCelloScope</td><td>BioSense Solution ApS</td><td></td><td>Optical camera</td></tr><tr><td>UniExplorer software&amp;nbsp;</td><td>BioSense Solution ApS</td><td></td><td>(8.0.0.7144 (RL3))</td></tr><tr><td>HaCaT cell</td><td></td><td></td><td>Donated from Bispebjerg Hospital</td></tr><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td>Medium for HaCaT</td></tr><tr><td>FBS</td><td>Gibco</td><td>10270</td><td>Fetal bovine serum</td></tr><tr><td>PBS</td><td>Gibco</td><td>D837</td><td>Without Mg+ and Ca2+</td></tr><tr><td>Pencillin-Streptomycin</td><td>Sigma</td><td>P0781</td><td>Antibiotics</td></tr><tr><td>Trypsin (10x)</td><td>Sigma</td><td>T3924</td><td></td></tr><tr><td>Mitomycin C</td><td>Tocris</td><td>3258</td><td>Inhibits proliferation</td></tr><tr><td>Microtiter plate</td><td>Greiner Bio-one</td><td>677 180</td><td></td></tr><tr><td>Incubator</td><td>Panasonic</td><td></td><td>37&amp;deg;C, 5% CO2</td></tr><tr><td>Hemocytometer</td><td>Assistent</td><td></td><td>T&amp;uuml;rk (0.1 mm)</td></tr><tr><td>Pipet boy</td><td>Accu-Jet pro</td><td></td><td></td></tr></tbody></table>

optimized scratch assay for in vitro testing of cell migration with an automated optical camera

细胞迁移是一个重要的过程, 影响健康的许多方面, 如伤口愈合和癌症, 因此, 这是至关重要的发展方法, 研究的迁移。由于其成本低、操作简单, 在体外检测化合物的抗性和亲迁移性方面一直是最常用的方法。然而, 一个经常报告的问题的检测是积累的细胞跨越边缘的划痕。此外, 为了从化验中获取数据, 不同曝光的图像必须在同一地点拍摄一段时间, 以比较迁移的运动。不同的分析程序可以用来描述的划痕闭合, 但他们是劳动密集型, 不准确, 和力量循环的温度变化。在这项研究中, 我们展示了一个优化的方法来测试的迁移效果,如自然发生的蛋白质人-和牛乳铁蛋白及其 n-末端肽乳铁素在上皮细胞系 HaCaT。一个关键的优化是清洗和刮在 PBS, 这消除了前面的细胞积累沿边缘。这可以通过去除阳离子来解释, 这已经被证明对角质形成细胞的连接有影响。为确保对迁移的真实检测, 在该协议中加入了丝裂霉素 C (DNA 合成抑制剂) 的预处理。最后, 我们演示了自动光学相机, 它消除了过度的温度循环, 手工劳动与划痕分析, 同时提高重现性和确保分析的相同部分的划痕随着时间的推移。

HaCaT 是角质细胞系, 是皮肤中最丰富的细胞类型之一。当伤口发生时, 皮肤细胞开始增殖并迁移到伤口床上以闭合伤口 (图 3)。因此, 这两个过程对正确愈合至关重要。
光学摄像头可以实时跟踪这两个过程。对于迁移, 系统提供三个数据集: 单元格覆盖区域、间隙宽度和间隙区域。当监测用乳铁蛋白、乳铁?...

Watch this Scientific Journal Video about Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera at JoVE.com

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

在这里, 我们描述了一个程序, 以测试的细胞迁移体外与自动光学相机显微镜。刮伤试验已被广泛使用, 在结束后的划痕是一个设定的周期。光学显微镜能够可靠、廉价地检测细胞的迁移。

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

optimized-scratch-assay-for-vitro-testing-cell-migration-with-an

Research

JoVE Journal

Biology

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

22.9K 次观看.  Roskilde University. 在这里, 我们描述了一个程序, 以测试的细胞迁移体外与自动光学相机显微镜。刮伤试验已被广泛使用, 在结束后的划痕是一个设定的周期。光学显微镜能够可靠、廉价地检测细胞的迁移。

视频: Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

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

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 ...

发育生物学

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 &#956;M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

This study focuses on an in vitro model of wound healing (scratch assay) as a mechanism for determining how environmental contaminants such as arsenic influence cellular migration. The results demonstrate that this in vitro assay provides rapid and early indications of changes to cellular migration prior to in vivo experimentation.

在体外显示砷对皮肤细胞迁移影响的划痕测定

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

环境科学

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 ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

医学

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

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

阐明 T 细胞动力学:实时迁移分析方法

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

作者聚焦:通过实时分析 T 细胞迁移了解疾病机制

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

原代小鼠 CD8+ T 细胞的实时体外迁移测定

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

用自动光学相机进行细胞迁移的体外试验优化划痕试验

摘要

探索更多视频

此视频中的章节

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

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

使用自动活单元成像仪的简单迁移/入侵工作流

在体外显示砷对皮肤细胞迁移影响的划痕测定

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

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

在体外细胞迁移和侵袭实验

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

原代小鼠 CD8+ T 细胞的实时体外迁移测定

原代小鼠 CD8+ T 细胞的实时体外迁移测定