作者希望感谢 Bhagawat 萨勃拉曼尼亚, 伊 (杰森), 保罗 Randazzo 和卡罗尔的父母阅读和评论的手稿。这项工作得到了国立卫生研究院国家癌症研究所的校内研究项目的支持。

1. 盘子的涂层 (1 天)
注意: 在处理时, 请务必不要将指纹或污垢留在盘子底部。
<ol><li>解冻小鼠胶原 iv 在冰上, 和稀释它与50毫米 HCl (pH 1.3) 准备3毫升的10µg/毫升胶原蛋白 iv 溶液。 注: 胶原蛋白在37° c 时会沉淀。因此, 保持胶原蛋白溶液在低温下融化并与之一起工作是很重要的。避免重复的冻融循环, 准备适当大小的等分和存储在-80 ° c。</li>
	<li>添加250µL 胶原 IV 溶液, 每井的12井玻璃底板, 并把它们放在一个适当大小, 紧合塑料盒。将湿纸巾放在盘子周围, 制造一个潮湿的房间并关上盒子。在室温下一夜之间孵育盘子。 注: 由于只有生长区域需要涂敷, 所以只有覆盖的盖子滑动, 这是连接到板的底部, 与胶原蛋白溶液 (请参见图 1a, B的示意图板)。</li>
	<li>第二天早上, 冲洗盘子与去离子 H2O 两次去除不吸收的胶原蛋白和缓冲。将水直接放到板的边缘, 而不是由板底和片形成的边缘 (如果水被 pipetted 到这个区域就会飞溅)。空气干燥板在层流罩。立即用盘子或在4° c 贮存5天。 注: 不同的细胞系可能需要不同的涂层, 如纤维连接蛋白或胶原蛋白 i。</li>
</ol>2. 电镀点检测 (2 天)
<ol><li>
准备细胞悬浮
	<ol>
<li>要准备细胞悬浮, 使用在 DMEM/F12 培养基中生长的60毫米组织培养皿中的细胞, 辅以5% 匹马血清至80% 汇合</li>
		<li>用钙和镁 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 冲洗细胞一次, 增加500µL 胰蛋白酶-EDTA (室温), 并孵育细胞为 3-5 分钟在一个孵化器在37° c, 5% CO2, 湿润的气氛。</li>
		<li>悬浮在4.5 毫升培养基中的分离细胞停止胰蛋白酶活性和计数20µL 分的细胞悬浮在例。</li>
		<li>将剩余的细胞在15毫升锥形管中通过离心 200 x g 进行3分钟的颗粒, 吸取上清液, 并重培养基中的细胞到 3 x 106细胞/毫升。 注: 对于其他涂层基底和/或细胞系, 最佳的细胞密度必须建立在稀释系列。建议将 3 x 106单元/mL 作为起始点。</li>
	</ol>
</li>
	<li>
电镀电池
	<ol>
<li>将10µL 的细胞悬浮液放在 IV 型胶原涂层12井玻璃底板的每个盖板的中心, 而不触及或刮伤涂层 (图 1a, B)。孵育板为30分钟, 在37° c, 湿润的气氛, 让细胞附着。在倒置显微镜中检查细胞的附着。 注意: 这在下落的边缘可以是最好看见的, 在那里单层的形成可以被观察 (图 1C)。如有必要, 可将钢板孵育至 3 h. 确保孵化箱内的湿度足够高, 因为水滴可以快速干燥。如果在这一步的下降量减少观察, 增加 PBS 的空间之间的井, 以增加湿度。</li>
		<li>用1毫升培养基轻轻冲洗两次, 去除非附着细胞。每井添加1毫升培养基。检查一个倒置的显微镜, 没有漂浮的细胞保持, 否则洗井再次。在37° c 的夜间孵育细胞, 5% CO2, 湿润的气氛, 获得良好定义的细胞表。 注: 漂浮细胞很可能会重新附着在盘子上, 形成不受欢迎的细胞群。</li>
	</ol>
</li>
</ol>3. 刺激细胞 (3 天)
<ol><li>使用反显微镜检查所有水井, 确保细胞菌落生长正常。如有必要, 标记不合适的水井, 不要使用。</li>
	<li>为所调查的每个条件适当地标记每一个板块。每一个条件运行一式两份或一式三份。</li>
	<li>改变培养基, 使其在细胞被刺激之前, 在含有0.1% 马血清 (饥饿培养基) 的 DMEM/F12 3 小时内使细胞挨饿。</li>
	<li>通过添加 EGF (5 ng/毫升最终浓度) 或其他所需的介质来刺激细胞, 用1毫升的 micropipettor 或旋转盘子轻轻地混合培养基。</li>
	<li>孵育该板块 1-4 天, 以观察边缘位移和边缘轮廓, 如第4.1 节所述, 或执行时间推移成像, 如在4.2 节所述。</li>
</ol>4. 细胞菌落和细胞迁移的可视化
<ol><li>
苏木精-曙红 (he) 染色测量菌落生长 (日 4-7)
	<ol>
<li>将70% 乙醇 (1 毫升/井) 的细胞固定在2分钟。</li>
		<li>染色核与苏木精 (1 毫升/井), 〜2分钟。</li>
		<li>用自来水冲洗细胞 (1 毫升/井) 直到细胞核是蓝色的, 5-10 分钟。</li>
		<li>染色细胞质与曙红 (1 毫升/井), 〜2分钟。</li>
		<li>用去离子水冲洗 (1 毫升/井)。 注意: 苏木精和红素溶液可以收集和使用多次, 直到染色变得微弱。</li>
		<li>空气干燥板。</li>
		<li>翻转板, 用尺子测量圆点的直径。或者, 用相机拍摄底片, 并分析 ImageJ 中的圆点直径。</li>
	</ol>
</li>
	<li>
延时显微镜 (3 天)
	<ol>
<li>打开孵化器显微镜, 将温度调整到37° c。用 dH 填充加湿器2o 将 CO2调整为5%。确保孵化室的湿化和机动阶段可以自由移动。关闭孵化室, 并允许显微镜调整到37° c。 注: 在这里使用的显微镜 (见材料表) 应加热到37° c, 至少3小时, 然后细胞被成像, 以避免漂移的焦点。</li>
		<li>把盘子放在孵化器显微镜的舞台上, 并设置舞台列表。图像两个对立的边缘和每个单元格点的中心 (图 1A)。</li>
		<li>使用10X 目标, 每3分钟拍摄一次图像, 15-24 小时。下载数据并在选择的程序中进行图像分析,例如、ImageJ 或 Matlab。</li>
	</ol>
</li>
</ol>

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作者没有什么要透露的。

在肿瘤进展过程中, 细胞从原发组织迁移到周围组织, 转移到远处的部位15。细胞群的迁移可以在点检测中观察到。在这里, 通过分析人乳腺癌细胞在 EGF 中的迁移表型来说明点测定。
为了获得准确和可复制的结果, 在点分析的设定中的几个步骤是至关重要的。首先, 板的涂层决定是否和如何强细胞能坚持板和如果细胞迁移。应注意的是, 该板的涂层与胶原 IV 或...

迁移分析被广泛用于评价肿瘤细胞的侵袭和转移潜能体外。最常见的情况是, 伤口或划痕检测是用来评估上皮细胞的迁移到一个单元清除区域1,2,3。为了进行划痕分析, 细胞被镀成单层, "划痕" 或无细胞区是用吸管尖端创建的。刮痕分析很容易建立与常用的组织培养用品, 可以进行多井板, 允许处理多个样品。然而, 当划痕时, 细胞从单层中被物理地移除, 并经常经历细胞死亡。此外, 在刮伤过程中, 细胞外基质常被破坏。同样, 使用硅刀片 (如信息处理室4) 或模具的5、6、7可以导致细胞的机械破坏和用于涂层的基质蛋白的部分去除。板.检测伤口或划痕闭合的另一个缺点是其有限的时间过程, 因为只有在抓痕闭合时才能分析细胞迁移。
在进行点检测时, 细胞被镀成一个圆形的菌落到涂层或未涂覆的平板上8。这种电镀策略的基本原理是获得具有定义边缘的细胞板, 它可以迁移或侵入周围的无细胞区, 而不会通过去除细胞或插入物而扰乱培养。斑点法的总体目标是观察细胞板的迁移, 如边缘位移或菌落直径, 以及执行时间推移成像, 以分析更高的时空分辨率的细胞移行表型。
细胞迁移可能受到各种微的影响, 如趋化因子, 细胞因子, 和生长因子, 如 EGF。egf 是通过结合其受体、egf 受体9而施加其生物效应的生长因子, 并增加肿瘤细胞的侵袭和转移行为4、9、10。在这里, 点法是用来研究 EGF 刺激细胞迁移在一个人侵入, 肺菌落形成乳腺癌细胞系 (MCF10CA1a)8,11,12。

<table><tbody><tr><td>MCF10CA1a cells</td><td>Karmanos Research Institute</td><td>N/A</td><td>cells used for assay</td></tr><tr><td>mouse collagen IV</td><td>BD Biosciences</td><td>354233</td><td>used to coat plates</td></tr><tr><td>trypsin / EDTA</td><td>Thermo Fisher</td><td>25300054</td><td>used to remove cells from tissue culture plates</td></tr><tr><td>DPBS&amp;nbsp;</td><td>Mediatech</td><td>21-031-CV</td><td>used to wash cells</td></tr><tr><td>DMEM / F12</td><td>Thermo Fisher</td><td>11320082</td><td>used as culture medium</td></tr><tr><td>horse serum</td><td>Thermo Fisher</td><td>26050088</td><td>used to supplement culture medium</td></tr><tr><td>12 well glass bottom plate</td><td>MatTek</td><td>P12G-1.5-14-F</td><td>used to grow cells for imaging</td></tr><tr><td>EGF (epidermal growth factor, human recombinant)</td><td>Peprotech</td><td>AF-100-15</td><td>used to stimulate cells</td></tr><tr><td>fatty acid free BSA</td><td>Sigma</td><td>A8806-1G</td><td>used to make buffer for EGF</td></tr><tr><td>hematoxylin</td><td>Sigma</td><td>HHS32</td><td>used to stain colonies</td></tr><tr><td>eosin</td><td>Electron Microscopy Sciences</td><td>26051-10</td><td>used to stain colonies</td></tr><tr><td>37 &amp;#176;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Sanyo</td><td>model MCO-18AIC (UV)</td><td></td></tr><tr><td>Zeiss AxioObserver with incubator chamber and motorized stage</td><td>Zeiss</td><td>model Axio Observer.Z1</td><td>used for time lapse imaging</td></tr><tr><td>ORCA Flash LT sCMOS camera</td><td>BioVision</td><td>C11440-42U-KIT</td><td>used for timelapse imaging in combination with Zeiss AxioObserver</td></tr><tr><td>Metamorph</td><td>BioVision</td><td>MMACQMIC</td><td>software used for time lapse imaging</td></tr><tr><td>inverted microscope</td><td>Olympus</td><td>model IX70</td><td>used to count cells and to check colonies in tissue culture</td></tr><tr><td>plastic box</td><td>Lock&amp;amp;Lock</td><td>N/A</td><td>used as humid chamber (any tight closing plastic box that fits the plates can be used)</td></tr></tbody></table>

using the dot assay to analyze migration of cell sheets

虽然复杂的有机体出现静态, 他们的组织是在连续的营业额之下。随着细胞衰老, 死亡, 并被新的细胞取代, 细胞在组织内以一种严密的方式移动。在肿瘤的发展过程中, 这种平衡受到干扰, 肿瘤细胞离开原发细胞侵入局部微环境, 前往远处的地点, 最终形成远处的转移性肿瘤。点法是一个简单的, 二维无约束迁移分析, 以评估细胞板的净移动到一个无细胞的区域, 并利用时间推移成像的细胞迁移参数。在这里, 点化验显示使用一个人侵入, 肺殖民地形成乳腺癌细胞线, MCF10CA1a, 以分析细胞的迁移反应表皮生长因子 (EGF), 这是已知的增加恶性电位的乳腺癌细胞和改变细胞的迁移表型。

这里提出的点检测 (图 1) 是利用侵入性, 肺菌落形成乳腺癌细胞 (MCF10CA1a) 作为一个模型系统进行的。点检测产生高度可重现的细胞点, 甚至在初学者的手, 使其方便, 易于执行的检测 (图 1D)。斑点分析结合 he 染色, 或时间推移成像和后续粒子图像测速 (PIV), 允许研究各种迁移参数, 包括上皮边缘的位移, 细胞速度和细?...

Watch this Scientific Journal Video about Using the Dot Assay to Analyze Migration of Cell Sheets at JoVE.com

Using the Dot Assay to Analyze Migration of Cell Sheets

用点法分析细胞板的迁移

细胞迁移是发展, 组织维护和修复, 和肿瘤发生的关键, 并由生长因子, 趋化因子, 和细胞因子调节。本协议描述的点法, 一个二维, 无约束的迁移分析, 以评估微提示的反应, 连接的, 有凝聚力的细胞表移表型。

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

using-the-dot-assay-to-analyze-migration-of-cell-sheets

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

6.8K 次观看.  National Institutes of Health. 细胞迁移是发展, 组织维护和修复, 和肿瘤发生的关键, 并由生长因子, 趋化因子, 和细胞因子调节。本协议描述的点法, 一个二维, 无约束的迁移分析, 以评估微提示的反应, 连接的, 有凝聚力的细胞表移表型。

视频: Using the Dot Assay to Analyze Migration of Cell Sheets

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

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 ...

科研

发育生物学

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 ...

癌症研究

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 ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

生物工程

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

从2DE凝胶点在蛋白质功能:在课慢性淋巴细胞白血病学到HS1

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

医学

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

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

量化颗粒水凝胶生物材料内部和颗粒状水凝胶生物材料中的三维细胞迁移

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

定制室测定细胞迁移

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

生物学

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

用于评估细胞运动和相关信号事件的机械控制的单细胞Durotaxis测定

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

The Transwell Migration Assay

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer. To quantify cell migration, a simple assay was developed in 1961 by Dr. Stephen Boyden, which is now known as the transwell migration assay or Boyden chamber assay. This set-up consists an insert which separates the wells of a multiwell plate into top and bottom compartments. Cells whose migration is to be studied are seeded into the top compartment and the chemoattractant solution is placed in the bottom compartment. After incubation, counting the cells in the bottom compartment allows quantification of migration induced by chemoattractants.
This video will review the commonly used experimental set-up for cell migration studies. Then we'll highlight a few key considerations, and outline a generalized protocol for running an experiment involving adherent cells. Lastly, we'll review various adaptations of this set-up currently being used to study different factors that affect migration.

The Transwell Migration Assay and Chemo-attractants

万士隆迁移实验

Cells migration in response to chemical cues is crucial to development, immunity and disease states such as cancer.  To quantify cell migration, a simple ...