Name: optimization of the wound scratch assay to detect changes in murine mesenchymal stromal cell migration after damage by soluble cigarette smoke extract
Uploaded: 2015-12-03T10:15:00
Description: Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

We are grateful to the other members of the lab for their technical assistance in developing the CSE damage assay, and their support and advice during the development of this assay. In addition, we are grateful to the Holy Cross Fenwick Scholar Committee for their support of Nicholas Cormier, and the Holy Cross College Honors program for their support of Alexander Yeo.

注意：对于该研究中，肺间质基质细胞（LR-的MSC）从远端肺组织中分离或者基于它们的细胞表面标记物的表达（CD45 阴性 ，CD31 阴性 ，的Sca-1的高，EPCAM 负）14,15，用植出生长16，或使用酶消化17。粘附LR-的MSCs培养在DMEM高糖和无谷氨酰胺，15％FBS，1×抗生素/抗 ​​真菌剂，和2mM谷氨酰胺（此后完全培养基）并在37℃，5％的CO 2。 LR-MSCs的传代...

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这里描述的协议提供了一个强大的定量，标准化的方法来执行和分析划痕试验。简单划痕测定法常规使用的在研究的许多不同领域，研究细胞迁移。然而，传统上的划痕试验一直缺乏标准化设置和定量协议，这已导致问题重复性7,10。许多的修改和优化用于提高划痕试验的减少这些问题，包括个人的活细胞成像，标准化塞为基础的测定，和三维微柱测定-4,8,10。但是，这些测定法可?...

细胞迁移是高度协调和至关重要的许多生理过程，如组织发育，修复和再生，以及病理过程，如癌症转移和动脉硬化1。细胞迁移的理解是特别相关的用于修复受损的组织和治疗的病理状态，包括细胞移植技术和人工组织嫁接2新兴疗法。鉴于间充质干细胞（MSCs）在介导组织修复3，用分析是严格计量的，适应性强，具有成本效益的感兴趣量化这些细胞的迁移能力的作用，目前的利息。重要的是，这样的测定必须是足够敏感的损害后，以检测在细胞迁移能力相对微妙的变化。目前的方法用于定量细胞的迁移，包括刮测定法，反式井迁移测定（Boyden小通道琥珀），微柱阵列，和细胞排除区检测具有一系列在再现性，可定制，量化，和成本效益-1,4,5-限制。此处所描述的优化划痕试验表明健壮的结果，量化的和基于图像的分析能力，成本效益，并适应其它应用。 划痕试验已被用于多种能力来评估不同的实验条件下5细胞迁移和增殖。该检测限嗣继承播种指定的细胞，他们成长，完成或接近汇合，和刮擦产生的单层用消毒针头或吸管尖6。是最常见的，在随机选择的位置7-9比较所述划痕在多个时间点的宽度进行分析。尽管其突出使用，划痕试验通过与重现性和定量的问题混淆。变异代划痕不仅改变细胞的微环境，而且还可以通过破坏该板面和底层的细胞外基质5阻碍细胞的迁移。测定是经常进行了超过7〜12小时，但对于细胞株显示较慢的迁移和更长的实验时间，增殖成为一个混杂变量7,10。最后，通过划痕过程中产生的衰老细胞中可以释放与缩小差距，在单层1所需的细胞外信令干扰因素。优化划痕测定需要创建一个一致的间隙不与表面性质干扰，减少分析时间长度，并防止操作过程中不希望的细胞死亡。止动基础的分析是小区排除区域测定的优化。该测定法利用塞子放置在排除细胞生长的孔的中间，但是允许细胞可围绕中心隔离区电镀。为了评估迁移，挡块被删除，并将所得排除区提供了用于迁移发生的表面。然而，该测定是困难的定制或调整10和对于一些应用，这种技术也可以成本过高。 在对比划痕测定和它们的衍生物，反-井迁移测定（或Boyden小室测定法）通过定量细胞移动从一个腔室的数目，通过微孔滤膜过滤，到含有趋化剂8,11的室评估迁移 ， 12。这种技术具有有限的效用为贴壁细胞如间充质干因为以下通过多孔膜的迁移，细胞粘附在膜侧暴露于趋化剂，并且可能很难准确量化。虽然检测是能够研究一些三维的迁移模式，受限制的细 ​​胞类型，这就是它能够准确量化细胞迁移限制其效用10。另一替代划伤试验采用了微柱阵列，它通过一个三维空间使用细胞变形并迁移到数组作为替代的能力测量细胞运动。聚二甲基硅氧烷（PDMS）弹性体固化过精密模具和用臭氧处理和纤连蛋白产生均匀和不可降解的微环境。根据需要，以评估细胞的进入阵列 4的能力的微柱间距也可以变化。模具是通过硅晶片的深反应离子蚀刻创建以创建高宽比阵列 13的负版本。虽然试验是由它的可定制性加强，能够通过细胞迁移的直接可视化模型的三维迁移和分析，难以产生微柱阵列在经济上阻碍了它的广泛使用。 在本协议中所描述的优化划痕试验提供了一个高效，COS叔有效的制造方法，可以使用免费提供的软件进行分析一致划痕。而不是之前和细胞迁移后，整个从头做起简单的宽度测量，该软件能够使用户在迁移前后测定总划伤的地方。这个前进限制了试图确定何处划痕宽度测量应采取的问题，以及是否划痕的宽度是沿着它的长度是均匀的。此外，细胞数，细胞融合和类型和损伤造成对细胞的程度的谨慎的优化，以便进一步优化测定法进行了讨论。

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">DMEM (high glucose, no added glutamine)</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">31053-036</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fetal Bovine Serum, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3091002</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Antibiotic/antimycotic, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SV3007901</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:527px;">Glutamine, 200mM, Thermo Scientific HyClone</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3003401</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">TypeLE cell dissociation reagent</td>
			<td style="width:221px;">Life Technologies</td>
			<td style="width:88px;">12563-01</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Dulbecco&#39;s Phosphate Buffered Saline, Thermo Scientific HyClone&#160;</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">SH3002802</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Falcon standard 60mm tissue culture dishes</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td style="width:88px;">08-772B</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Fisherbrand Sterile 200ul pipet tips</td>
			<td style="width:221px;">Fisher Scientific</td>
			<td>02-707-502</td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Inverted light microscope with camera attachment</td>
			<td style="width:221px;">Variable</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:527px;">ImageJ software&#160;</td>
			<td style="width:221px;">http://imagej.nih.gov/ij/</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:527px;">MRI_Wound_Healting plugin</td>
			<td style="width:221px;"><a href="http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool">http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool</a></td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:527px;">Statistical analysis software</td>
			<td style="width:221px;">Microsoft Excel</td>
			<td style="width:88px;"></td>
			<td style="width:307px;"></td>
		</tr>
	</tbody>
</table>

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.

这里介绍的划痕试验用鼠肺间质居民基质细胞（LR-MSCS）进行，分离引用在协议中说明。在LR-MSCs的接种500,000个细胞在60毫米组织培养皿的密度，并在48小时生长至90％汇合。为了产生损害，4％CSE（如上所述）中的与细胞一起培养24小时后的初始播种期间，但在划痕试验前。对于每个测定，用200μl移液管尖端部中产生的划痕。最初的图像拍摄已产生的划痕后，任何松散的细胞和碎片已经被冲走完整的?...

Watch this Scientific Journal Video about Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract at JoVE

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

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

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Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

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

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

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

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

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Visualization of Tangential Cell Migration 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 ...