Name: a novel in vitro wound healing assay to evaluate cell migration
Uploaded: 2018-03-17T13:00:00
Description: Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

这项工作得到了 Sapienza 大学和意大利囊性纤维化研究基金会 (来自锡耶纳、Sondrio Valchiavenna、蜡样 Il Sorriso di 和帕维亚) 的 FFC#11/2014 代表团的资助。这项工作的一部分也得到了 FILAS 授权 FILAS-RU-2014-1020 的支持。
我们感谢费雷拉博士 (U.O.C. Genetica, 无依儿童 Giannina Gaslini, 意大利 Genova) 提供支气管上皮细胞。

1. 细胞制剂
<ol><li>种子 2.5x106细胞在10毫升的最低基本培养基 (内存) 补充2毫米谷氨酰胺 (MEMg), 加上10% 胎牛血清 (血清), 抗生素 (0.1 毫克/毫升青霉素和链霉素), 和嘌呤霉素 (0.5 µg/毫升的选择和维护细胞线) 在一个 T75 瓶。将烧瓶在37摄氏度和 5% CO2孵化2天。在开始实验之前, 检查细胞在倒置显微镜下的汇合。 注: 用于实验的细胞是永生化的人支气管上皮细胞转基因与慢病毒载体的?...

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+defective+cystic+fibrosis+airway+epithelial+wound+repair+after+CFTR+rescue.">Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue.</a> Eur Respir J. 40, 1390-1400 (2012).</li><li>Gan, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidermal+growth+factor+receptor+(EGFR)+tyrosine+kinase+inhibitor+AG1478+increases+the+formation+of+inactive+untethered+EGFR+dimers.+Implications+for+combination+therapy+with+monoclonal+antibody+806.">The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 increases the formation of inactive untethered EGFR dimers. Implications for combination therapy with monoclonal antibody 806.</a> J Biol Chem. 282, 2840-2850 (2007).</li><li>Tokumaru, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+keratinocyte+migration+via+transactivation+of+the+epidermal+growth+factor+receptor+by+the+antimicrobial+peptide+LL-37.">Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37.</a> J Immunol. 175, 4662-4668 (2005).</li><li>Tjabringa, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+peptide+LL-37+activates+innate+immunity+at+the+airway+epithelial+surface+by+transactivation+of+the+epidermal+growth+factor+receptor.">The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor.</a> J Immunol. 171, 6690-6696 (2003).</li><li>Di Grazia, A., Luca, V., Segev-Zarko, L. A., Shai, Y., Mangoni, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporins+A+and+B+stimulate+migration+of+HaCaT+keratinocytes+and+kill+intracellular+Staphylococcus+aureus.">Temporins A and B stimulate migration of HaCaT keratinocytes and kill intracellular Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 2520-2527 (2014).</li></ol>

细胞迁移是许多生理和病理事件中必不可少的过程, 包括伤口愈合、胚胎发育和肿瘤转移。研究细胞迁移的基本过程体外涉及: (一) 建立细胞单层, (ii) 在细胞的汇合层内产生伪伤口, (iii) 在达到伤口闭合之前, 在不同时间间隔捕获图像。, 并 (iv) 对图像序列进行分析, 以量化所选细胞的迁移速度。
研究结果表明, 培养基的应用是定量评价细胞迁移特性的客观可靠的实验技?...

众所周知, 动物创面愈合是重建损伤后组织层完整性和正常功能的一个基本过程1。尽管暴露于外部环境的上皮表面 (例如,皮肤、呼吸道和胃肠道) 构成了对物理和化学侮辱的保护性屏障, 但伤口的形成很容易发生, 特别是在手术或微生物感染2。例如, 由机会性细菌病原体 (特别是囊性纤维化) (CF) 引起的肺组织的殖民化, 导致呼吸道上皮损害, 从而造成呼吸衰竭3, 4。伤口愈合是一个复杂的主机修复机制, 恢复正常结构的受伤组织5。它的特点是初始炎症, 其次是再生期, 包括上皮, 血管生成和组织重塑与胶原蛋白生产和细胞分化6,7,8.为确保上皮完整性和控制微生物增殖, 所有生物体都产生防御分子, 包括抗菌肽 (安培)9,10。创伤愈合过程是非常困难的模拟在体外由于缺乏细胞碎片和复杂的相互作用在不同的细胞类型之间。然而, 肽的体外能力通过刺激上皮细胞的迁移来加速闭合假伤口, 这表明它有能力愈合受损的上皮。事实上, 细胞迁移是创伤愈合中的速率限制事件, 研究影响细胞迁移的因素将有助于改善伤口愈合的靶向治疗。
在此基础上, 提出了一种高度可重现性的实验检测方法, 它是基于特殊的硅胶培养插入来评估细胞迁移的体外。它是建立在一个500微米的缺口 (伪伤口) 的汇合细胞单层。在人工 "受伤" 领域边缘的细胞将开始迁移到无细胞区, 形成新的细胞细胞接触。文化插入代表了一个新的工具, 快速伤口愈合实验。提供两个由500微米壁隔开的储层, 它们可以适当地放置在3厘米厚的盘子里, 或者放在12井板的井中。将插入的每个隔间填满一个细胞悬浮, 允许细胞在每个指定区域内生长直至汇合, 而除去插入物将产生一个干净的无细胞间隙, 约500微米 (与隔离墙相同的宽度)。一个适当的细胞培养培养基, 辅以测试化合物, 然后可以添加到盘子里/好。然后, 在倒置显微镜下, 可以在不同的时间间隔内可视化间隙闭合, 最好是配备有摄像机进行图像采集。最后, 通过基于 web 的自动图像分析程序测量细胞覆盖区域的变化, 可以量化伤口闭合和细胞迁移的速度。总的来说, 这种方法是一个向前迈进的经典化验, 其中一个划痕是手动切割汇合细胞单膜与无菌针或吸管提示11。事实上, 最后的程序可以摧毁盘子的塑料底部/井和表面涂层, 造成皱纹。此外, "受伤" 区域没有一个明确的宽度沿整个长度的差距, 因为这高度取决于研究人员的压力, 对针/尖。此外, 脱落的细胞可以形成团簇的活和死细胞在边缘的划痕;此外, 将活细胞扩散到 "受伤" 区域可能会干扰细胞迁移速度, 产生不可重现的结果12。
此外, 由于一个划痕图像分析平台, 用户可以迅速收到 (几分钟内) 的数量数据的迁移行为的选定细胞, 而不需要获得额外的软件。该平台能够分析低 (~ 5 x)、中等 (~ 10 x) 和高 (~ 20 x) 放大倍数的相位对比显微镜图像。上传图像的 zip 文件后 (在 * 中. jpg, *. jp2, *. png, *. tiff 格式) 将自动进行分析, 以生成一个摘要文件, 显示单元格覆盖区域和划痕区域的百分比, 以及单元格的速度迁移, 在不同的时间间隔。
在这项工作中, 通过使用青蛙皮肤安培导数,即esc (1-21) 和它的 diastereomer esc (1-21)-1 c13, 和一个支气管细胞线表达功能 CF 跨膜电导调节器 (CFTR)14,15,以多肽诱导的细胞迁移为例, 与未处理的 (对照) 样品进行比较。注意, 气道上皮和 CFTR 在维持肺功能和伤口修复方面起着至关重要的作用16。此外, 通过选择性抑制剂 (例如, AG1478), 表皮生长因子受体 (EGFR) 的17 , 证据表明, 上述肽诱导的支气管细胞迁移涉及激活 EGFR12,报告了18 。
总之, 这个过程的目的是显示如何使用这种硅胶培养插入物代表一个快速和容易接近的检测, 以准确地确定黏附分子的迁移 (例如,支气管上皮细胞) 和控制此类事件的机制。

<table border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Minimal essential medium (MEM)&#160;</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB2071L</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glutamine</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECS0180DH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin and Streptomycin</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Puromycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin/EDTA 1X in PBS</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3052D</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">DPBS without calcium and magnesium (CMF-PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DPBS with calcium and magnesium (PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ibidi Culture-Insert 2 well</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">80209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Wimasis Image Analysis</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">30002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRISM software&#160;</td>
			<td>GraphPad</td>
			<td style="width:119px;">version 6.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro wound healing assay to evaluate cell migration

这项工作的目的是显示一种新的方法来评估一些免疫调节分子, 如抗菌肽 (安培) 的能力, 以刺激细胞迁移。重要的是, 细胞迁移是创伤愈合过程中的速率限制事件, 重建损伤后组织层的完整性和正常功能。这种方法的优势, 在经典的化验, 这是基于一个手工制造的从头在细胞单层, 是使用特殊的硅胶文化插入提供两个舱室, 以创建一个无细胞的伪伤口领域, 有明确的宽度 (500 微米).此外, 由于一个自动化的图像分析平台, 有可能迅速获得定量数据的伤口闭合和细胞迁移的速度。更确切地说, 两个蛙皮放大器对支气管上皮细胞迁移的影响将显示出来。此外, 预处理这些细胞与特定抑制剂将提供信息的分子机制的基础上, 这些事件。

本协议用于确定1-21 和 esc (1-21)-1 c 在表达功能 CFTR 的支气管上皮细胞的细胞迁移活动中的创面愈合效果。在这项试验中, 将培养物插入12井板的井中, 每个隔间都有3.5万个细胞 MEMg, 辅以10% 的血清。细胞在24小时之内到达了完全汇合, 然后产生了500微米的缝隙, 每个井都充满了含有不同浓度肽的 MEMg。实验重复了四次。如图 2所示, 在细胞单层中产生的伪?...

Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

在这里, 我们提出一个评估肽对支气管上皮细胞迁移的影响的协议。这种方法允许快速和高度重复获取的数量数据的速度的细胞迁移和伤口闭合。

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In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-GuÃƒÆ’Ã†'Ãƒâ€šÃ‚Â©rin

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Use of a Monocyte Monolayer Assay to Evaluate FcÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Receptor-mediated Phagocytosis

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...