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 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

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

16.8K 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

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

科研

发育生物学

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 ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

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

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

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

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

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

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

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

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

Here, we present a protocol to perform a semi-high throughput cell migration assay on a 96-well cell culture plate. This protocol is a fast, simple and economical method to create consistent scratch wounds on a cell monolayer.

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 ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

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

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

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

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

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 ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior 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 ...