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3-4次，以确保细胞群与用于迁移测定相对同质生长特性。 1.准备一个融合的单层<ol><li>分离从标准的100毫米培养皿中的MSC，洗涤细胞，用10毫升37℃的预热的PBS中，吸出PBS并加入胰蛋白酶4毫升（0.25％）。孵育在37℃培养箱中3-5分钟。吸的CELLS到锥形管中，以300g添加完全培养基，离心等体积为5分钟以沉淀下来的细胞。 注意:在胰蛋白酶长孵育可以改变细胞表面受体，并可能影响迁移。 </li><li>吸媒体和悬浮细胞，新鲜完整的媒体。计数细胞，用台盼蓝染色排除死细胞和板50万间充质干细胞在4毫升完整的媒体对60毫米的细胞培养皿。 注意:根据细胞的来源上，测试可能是必要的，以确定所需要的细胞的最佳数量。 </li><li>孵育细胞，直到板为90％融合了最佳的细胞附着和增殖，一般48-72小时的干细胞。 注:100％融合，不推荐，因为许多锚定依赖性细胞的体验接触抑制，这可能会改变他们的迁移能力。板的小于80％汇合，将导致不能使用的照片，因为图像分析软件将解释间隙细胞作为划痕的周边的部分之间秒（ 见图1D）。 </li><li>后的细胞达到90％汇合时，损伤细胞以适合应用。为了评估暴露于可溶性香烟烟雾提取物（CSE）后的细胞损伤，经到25 ml完全培养基在Buchner瓶超过2分钟，18吸引来自肯塔基大学的1个研究香烟（3R4F）烟雾产生100％的自定义搜索引擎。 </li><li>孵育细胞24小时4毫升1-4％CSE稀释在完全培养基中。培养后，用4毫升温无菌PBS洗细胞两次，用4ml新鲜完全培养基更换之前。 注:毁伤能力的细胞具有高浓度的香烟烟雾提取物（&gt; 5％），可大大降低汇合。 </li></ol> 2.创建暂存<ol><li>在一个无菌的环境，请从受损的干细胞的60毫米的钢板盖，并奠定了直边（例如无菌塑料尺）交流罗斯板的顶部边缘。如果不取出介质，使用消毒200μl枪头进行无菌直边引导，使整个板块差一分四点平行划痕大约间隔5毫米，50毫米长。 注:直边最小化所需要的板材，当它是在显微镜舞台上旋转的调整量。当务之急是划痕出现在屏幕上完全垂直（或水平）。在60毫米的钢板产生多个划痕不会改变，影响细胞迁移（数据未显示）的方式的微环境，但也可以是对其它细胞类型的考虑。使用更小的孔的不推荐，因为细胞生长接近井的边缘的变异性变得更加明显，并且可能影响均匀代划痕。 </li><li>吸媒体，轻轻用2ml预热完整的媒体，以防止细胞粘到划痕的中心洗平板，并以除去细胞s表示已松动的的刮伤。更换用4毫升新鲜完整的媒体。 </li><li>用无菌永久性标记，标号1-4在板的下侧的每个刮在不会与成像划痕干扰位置。 注意:从同一个临时图像迁移前后进行比较是很重要的，因为可以有变化的划痕宽度。 </li></ol> 3.以暂存的初始图像注意:如果划痕试验是在多个板中进行，所以建议在单个板的所有划痕初始图像被拍摄之前划痕上的下一个板制成。 <ol><li>使用倒置相差显微镜用照相机以生成分析所需的图像。 </li><li>定位在显微镜的培养板，并使用低功率物镜（10X）来定位刮＃1。如果缩合在培养板的盖子妨碍清晰IMAGE中的细胞，用加热的阶段设定为37℃。 </li><li>保持刮完全水平和在视场的中心，得到尽可能多的图像作为必要在包括90％的划痕长度的。获得的耐擦伤不同的部分图像的图像之间没有重叠。 注意:光在培养皿的边缘上的折射曝光过度的图像，使它们不可能分析。因此，不从第一个拍摄图像和最后划痕的长度的5％。 </li><li>保存每个从无到有的图像在一个单独的文件夹中。 </li><li>重复步骤3.2-3.4是否有划痕2，3和4。 </li><li>最初的影像后立即返回板的孵化器。让细胞迁移4-7小时。 注:评估干细胞受损与CSE时7小时是最佳的。孵化细胞大于7小时不建议，因为细胞的增殖可以作为细胞迁移混杂变量（参见下面的可选注8.2）。 </li></ol><p类="jove_title"&gt; 4。初步分析划痕图片<ol><li> ImageJ的下载和安装伤口愈合工具插件（请参阅的材料/设备表）。 </li><li>在打开ImageJ的图像文件，然后单击"进程"和"查找边缘"突出周边划伤的伤口愈合工具来计算临时区域（ 图2B）的细胞。接下来，单击"图片"，"调整"，"颜色阈值"，并在标有"门槛颜色"下拉菜单值更改为"B＆W"。 </li><li>在"颜色阈值"菜单，同时调整亮度阈值滑块，直到从无到有，小区前的最佳的对比度达到。要允许尝试创建图像的对比度，完全移动底部滑块向右（全黑图像），然后调整顶部滑块产生最大对比度时，最大的范围内。 </li><li>慢慢调整前BRIghtness滑块从左至右直到图像干净地显示划痕（图2C）的轮廓。一旦划伤的边缘已经确定，单击"更多选项"，在ImageJ的工具栏，在下拉菜单中选择"MRI_Wound_Healing_Tool"，然后按"M"键（将出现在ImageJ的工具栏）突出刮开区并输出所计算的区域中一个结果弹出出窗口（图2D）。 </li><li>复制并从结果窗口 （图2E）的所有号码粘贴到Excel电子表格。请务必将数据从单独划痕分开。 </li></ol> 5.与划痕的最终图像<ol><li>在细胞迁移（4-7小时），重复步骤3.2-3.4。图像以相同的顺序它们被划伤，使细胞在每个板上有相等的时间迁移的板。 注:细胞可以在多个时间点进行成像，这取决于它们的森西拉了一到温度和pH值的变化。或者，活细胞成像可以用于跟踪单元的实时，因为它们迁移。 </li></ol> 6.分析最终划痕图片<ol><li>最终划痕图像重复步骤4.2-4.5。 </li></ol> 7.计算刮开区量化迁移<ol><li>通过平均各刮痕的初始面积值计算的平均预迁移区域。 </li><li>减去最后的迁移区域的每个图像，在步骤6中计算出的，从平均迁移前区域（步骤7.1）计算迁移的总量。生成值的每一个从无到有代表迁移变化的列表。 </li><li>池相同的测试组（ 如对细胞的所有板和所有的划痕暴露在0％CSE）内多处抓痕的数据。使用方差测试的分析方法来分析实验组之间的差异。 </li><li>如果细胞迁移慢并且需要较长的培育时间划伤（大于10〜12小时）后，执行的BrdU或EDU掺入法19，以确定是否细胞中，其中移民法正在发生期间发生的任何扩散。选择迁移实验时间，以避免显著细胞增殖。 </li></ol> 8.可选<ol><li>为了评估显著细胞衰老是否发生如划痕过程的结果，搔抓19之后执行在测试板上的衰老相关的β-半乳糖苷酶测定在预定的时间。 </li></ol>

<ol>
	<li>Gough, W., Hulkower, K. I., Lynch, R., 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=A+quantitative,+facile,+and+high-throughput+image-based+cell+migration+method+is+a+robust+alternative+to+the+scratch+assay.">A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay.</a> J. Biomol. Screen. 16 (2), 155-163 (2011).</li><li>Ridley, A. J., Schwartz, M. A., Burridge, 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=Cell+migration:+integrating+signals+from+front+to+back.">Cell migration: integrating signals from front to back.</a> Science. 302 (5651), 1704-1709 (2013).</li><li>Sharma, R. R., Pollock, K., Hubel, A., McKenna, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+or+stromal+cells:+a+review+of+clinical+applications+and+manufacturing+practices.">Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices.</a> Transfusion. 54 (5), 1418-1437 (2014).</li><li>Booth-Gauthier, E. A., Du, V., Ghibaudo, M., 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=Hutchinson-Gilford+progeria+syndrome+alters+nuclear+shape+and+reduces+cell+motility+in+three+dimensional+model+substrates.">Hutchinson-Gilford progeria syndrome alters nuclear shape and reduces cell motility in three dimensional model substrates.</a> Integr. Biol. (Camb). 5 (3), 569-577 (2013).</li><li>Keese, C. R., Wegener, J., Walker, S. R., Giaever, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+wound-healing+assay+for+cells+in+vitro.">Electrical wound-healing assay for cells in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (6), 1554-1559 (2004).</li><li>Pinco, K. A., He, W., Yang, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha4beta1+Integrin+Regulates+Lamellipodia+Protrusion+Via+a+Focal+Complex/focal+Adhesion-Independent+Mechanism.">Alpha4beta1 Integrin Regulates Lamellipodia Protrusion Via a Focal Complex/focal Adhesion-Independent Mechanism.</a> Mol. Biol. Cell. 13 (9), 3203-3217 (2002).</li><li>Fronza, M., Heinzmann, B., Hamburger, M., Laufer, S., Merfort, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+wound+healing+effect+of+Calendula+extracts+using+the+scratch+assay+with+3T3+fibroblasts.">Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts.</a> J. Ethnopharmacol. 126 (3), 463-467 (2009).</li><li>Justus, C. R., Leffler, N., Ruiz-Echevarria, M., Yang, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> J. Vis. Exp. (88), (2014).</li><li>Walter, M. N., Wright, K. T., Fuller, H. R., MacNeil, S., Johnson, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-conditioned+medium+accelerates+skin+wound+healing:+an+in+vitro+study+of+fibroblast+and+keratinocyte+scratch+assays.">Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays.</a> Exp. Cell Res. 316 (7), 1271-1281 (2010).</li><li>Goetsch, K. P., Niesler, C. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+scratch+assay+for+in+vitro+skeletal+muscle+wound+healing+analysis.">Optimization of the scratch assay for in vitro skeletal muscle wound healing analysis.</a> Anal. Biochem. 411 (1), 158-160 (2011).</li><li>Chung, C. A., Chen, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cell+sedimentation+on+measuring+chondrocyte+population+migration+using+a+Boyden+chamber.">The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber.</a> J. Theor. Biol. 261 (4), 610-625 (2009).</li><li>Fabbri, M., Bianchi, E., Fumagalli, L., Pardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lymphocyte+traffic+by+adhesion+molecules.">Regulation of lymphocyte traffic by adhesion molecules.</a> Inflamm. Res. 48 (5), 239-246 (1999).</li><li>Saez, A., Ghibaudo, M., Buguin, A., Silberzan, P., Ladoux, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rigidity-driven+growth+and+migration+of+epithelial+cells+on+microstructured+anisotropic+substrates.">Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (20), 8281-8286 (2007).</li><li>Paxson, J. A., Gruntman, A. M., Davis, A. M., 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=Age+Dependence+of+Lung+Mesenchymal+Stromal+Cell+Dynamics+Following+Pneumonectomy.">Age Dependence of Lung Mesenchymal Stromal Cell Dynamics Following Pneumonectomy.</a> Stem Cells Dev. 22 (24), 3214-3225 (2013).</li><li>McQualter, J. L., Brouard, N., Williams, B., 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=Endogenous+fibroblastic+progenitor+cells+in+the+adult+mouse+lung+are+highly+enriched+in+the+sca-1+positive+cell+fraction.">Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction.</a> Stem Cells. 27 (3), 623-633 (2009).</li><li>Hoffman, A. M., Paxson, J. A., Mazan, M. R., 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=Lung-Derived+Mesenchymal+Stromal+Cell+Post-Transplantation+Survival,+Persistence,+Paracrine+Expression,+and+Repair+of+Elastase-Injured+Lung.">Lung-Derived Mesenchymal Stromal Cell Post-Transplantation Survival, Persistence, Paracrine Expression, and Repair of Elastase-Injured Lung.</a> Stem Cells Dev. 20 (10), 1779-1792 (2011).</li><li>Beane, O. S., Fonseca, V. C., Cooper, L. L., Koren, G., Darling, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+aging+on+the+regenerative+properties+of+bone+marrow-,+muscle-,+and+adipose-derived+mesenchymal+stem/stromal+cells.">Impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells.</a> PLoS One. 9 (12), e115963 (2014).</li><li>Chen, L., Ge, Q., Tjin, G., 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=Effects+of+cigarette+smoke+extract+on+human+airway+smooth+muscle+cells+in+COPD.">Effects of cigarette smoke extract on human airway smooth muscle cells in COPD.</a> Eur. Respir. J. 44 (3), 634-646 (2014).</li><li>Itahana, K., Campisi, J., Dimri, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+detect+biomarkers+of+cellular+senescence:+the+senescence-associated+beta-galactosidase+assay.">Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase assay.</a> Methods Mol. Biol. 371, 21-31 (2007).</li></ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>DMEM (high glucose, no added glutamine)</td><td>Life Technologies</td><td>31053-036</td><td></td></tr><tr><td>Fetal Bovine Serum, Thermo Scientific HyClone</td><td>Fisher Scientific</td><td>SH3091002</td><td></td></tr><tr><td>Antibiotic/antimycotic, Thermo Scientific HyClone&amp;nbsp;</td><td>Fisher Scientific</td><td>SV3007901</td><td></td></tr><tr><td>Glutamine, 200 mM, Thermo Scientific HyClone</td><td>Fisher Scientific</td><td>SH3003401</td><td></td></tr><tr><td>TypeLE cell dissociation reagent</td><td>Life Technologies</td><td>12563-01</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline, Thermo Scientific HyClone&amp;nbsp;</td><td>Fisher Scientific</td><td>SH3002802</td><td></td></tr><tr><td>Falcon standard 60 mm tissue culture dishes</td><td>Fisher Scientific</td><td>08-772B</td><td></td></tr><tr><td>Fisherbrand Sterile 200 &amp;mu;l pipet tips</td><td>Fisher Scientific</td><td>02-707-502</td><td></td></tr><tr><td>Inverted light microscope with camera attachment</td><td>Variable</td><td></td><td></td></tr><tr><td>ImageJ software&amp;nbsp;</td><td>http://imagej.nih.gov/ij/</td><td></td><td></td></tr><tr><td>MRI_Wound_Healing plugin</td><td>&lt;a href=&quot;http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool&quot;&gt;http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool&lt;/a&gt;</td><td></td><td></td></tr><tr><td>Statistical analysis software</td><td>Microsoft Excel</td><td></td><td></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

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

optimization-wound-scratch-assay-to-detect-changes-murine-mesenchymal

Research

JoVE Journal

Developmental Biology

16.9K 次观看.  College of the Holy Cross. 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.

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

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

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

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

免疫与感染

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

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 ...

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

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

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

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

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 ...

生物学

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

经济高效且适应性强的划痕迁移检测

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

使用划痕伤口迁移和球状体发芽测定法研究血管生成

Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets.
This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.

作者聚焦：通过检测开发中的挑战和创新研究血管生成

This study presents the two-dimensional (2D) scratch wound migration assay and the three-dimensional (3D) spheroid sprouting assay, along with their respective downstream analysis methods, including RNA extraction and immunocytochemistry, as suitable assays to study angiogenesis in vitro.

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A ...

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

摘要

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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

在体外显示砷对皮肤细胞迁移影响的划痕测定

一种新的体外创面愈合试验评价细胞迁移

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

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

用自动光学相机进行细胞迁移的体外试验优化划痕试验

经济高效且适应性强的划痕迁移检测

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成

利用划痕伤口迁移测定和球状体发芽测定在功能和分子水平上研究血管生成