我们要感谢实验室的老成员, 帮助改善和完善这些技术的实际状态: Dr. 西莉亚。Dr. 安娜 Mrowiec, Dr., 拉肯纳和 Dr.-加西亚。我们感谢医院 Clínico 体育馆圣女 Arrixaca 大力支持这些技术的发展。还有干杯卡洛斯三世, 基金 de 门多萨市 Sanitarias。计划国家 i + D + i 和干杯研究所-Subdirección 总 de Evaluación y 调值研究 (批准号: PI13/00794);www.isciii.es. Fondos 菲德 "manera de hacer 欧罗巴"。我们还感谢穆尔西亚、IMIB-Arrixaca 和 FFIS 的行政支助和援助。最后, 我们要特别感谢 Dr. 伊莎贝尔-马丁内斯-Argudo 和 Facultad de Ciencias Ambientales Bioquímica, 校园 Tecnológico de Fábrica 武器, 卡斯蒂利亚 la 恰尔, 托莱多为他们的亲切的支持自愿割让生物医学和生物技术实验室, 使本论文的拍摄部分成为可能。

1. 定量研究的人工伤口划痕法
<ol>
	<li>细胞单层制备
	<ol>
		<li>在无菌条件下工作, 用血清补充培养基在培养瓶中种 Mv1Lu 或 HaCaT 上皮细胞。每24-48 小时刷新一次介质。在单元格达到80% 汇合后, 使用适当的方法分离单元格,即, trypsinization9。 注: Mv1Lu 和 HaCaT 上皮细胞系分别用 EMEM 和 DEMEM 培养基培养, 并辅以2毫米 l-谷氨酰胺, 在37° c 下孵育 5% CO2的受控气氛。每一个细胞线必须进行彻底的检测, 以确定细胞浓度和时间需要达到完全70-100。通常为 Mv1Lu 或 HaCaT 细胞, 2-4 x 104或 4-6 x 104单元格/好, 分别在 175 cm2培养烧瓶中播种, 在80% 汇合处的收益率高达 35 x 106 Mv1Lu 或 1 x 107 HaCaT 单元格。</li>
		<li>在12井或24井培养板, 种子在每井2毫升的细胞。每24-48 小时刷新一次介质, 确保电池数量足够高, 可以在2或3天后达到100% 汇合。</li>
		<li>细胞到达汇合处后, 取出血清补充培养基, 用新鲜无血清培养基冲洗两次。在刮伤前24小时将细胞保持在无血清培养基中。 注: Mv1Lu 或 HaCaT 细胞没有特殊的承印物要求;然而, 对于易在无血清条件下自发分离的细胞系, 使用聚 l-赖氨酸溶液的培养板表面的预应考虑为10。</li>
	</ol>
	</li>
	<li>单层刮伤
	<ol>
		<li>在无菌环境中工作, 通过牢固地拖动20µL 或200µL 无菌微尖端的窄端, 根据所需的划痕宽度, 从头开始培养单层。保持尖端与培养面垂直, 最大限度地提高间隙均匀性。<ol>
			<li>将培养皿放置在深色表面上, 以清楚地监视细胞单分子。如果需要, 在每个井上执行两个垂直划痕 (十), 以研究多达4迁移区。</li>
		</ol>
		</li>
		<li>通过轻轻去除培养基, 轻轻地加入新鲜无血清培养基, 洗涤分离细胞。重复两次, 或直到大多数未连接的单元格被移除。</li>
	</ol>
	</li>
	<li>实验程序和数据收集
	<ol>
		<li>采用一台倒置的相衬显微镜, 结合 CCD 相机, 以每井的划痕间隙为中心的4前处理参考图像。通过将显微镜场的边缘与划痕的相邻交叉点对齐, 选择感兴趣区域。 注意: 通常, 图像是使用10x 放大倍数和 1280 x 1024 像素图像大小获取的。如上所述, 选择感兴趣的区域有助于方便地定位和验证每井4测量。</li>
		<li>一旦所有的图像获得, 指定治疗井;用含有选定处理的新鲜培养基在井中交换培养基。 注: 另外, 对于不干扰培养基同质性的化学品或化合物, 可以直接将治疗接种到培养基中。</li>
		<li>孵育刮板 (37 ° c 与控制气氛包含 5% CO2), 直到观察到的条件达到90% 划痕间隙闭合。避免完全关闭。 注: 总间隙闭合使后处理图片收集无法检测, 无法确定间隙闭合的时间基准。在 Mv1Lu 或 HaCaT 细胞的情况下, 需要16-19 小时达到90% 汇合。</li>
		<li>通过固定细胞来阻止细胞迁移;在 PBS 中用1毫升4% 福尔马林轻轻地取代实验培养基。在 RT 处孵育15分钟。</li>
		<li>清洗细胞去除过量福尔马林;用新的 PBS 将溶液轻轻地替换在井中。重复至少两次。 注: 在这个阶段, 在密封之后, 板材可以被存放在4° c 无限期地。</li>
		<li>从划痕后捕获的原始参考区域中对每一个井后的参考图像进行处理。使用相同的设备 (倒置光学相衬显微镜, 结合 CCD 相机) 和匹配的图片设置 (放大和数字分辨率/密度)。 注意: 对于单独迁移并填补间隙的单元格, 独立于相干锋的形成 (例如, MDA-MB-231 乳腺癌人细胞), 时效图像可能允许计算单个细胞迁移速度。</li>
	</ol>
	</li>
	<li>图像分析与迁移量化
	<ol>
		<li>使用图像处理软件 (例如, ImageJ), 定义划痕间隙限制, 并确定在预处理图片中多达四测量的间隙表面。将数据记录为 "预处理间隙曲面" (PREGAP)。对后处理图片重复相同的过程。将数据记录为 "后处理间隙曲面" (POSTGAP)。<ol>
			<li>使用 ImageJ 打开录制的图像。在 "图像" 菜单中, 将图像类型设置为8位。在 "进程" 菜单中, 转到 "过滤器" 子菜单并应用 "方差" 过滤。</li>
			<li>在 "图像" 菜单中, 输入 "调整" 子菜单, 并将阈值设置为黑白 (B 和 #38; W), 确保未选中 "暗背景"。在 "进程" 菜单中, 转到 "二进制" 子菜单, 然后选择 "填充孔"。</li>
			<li>在 "分析" 菜单中, 选择 "设置测量" 并激活 "区域"。然后绘制在迁移边缘轮廓之后的可测量区域, 从而划定间隙。</li>
			<li>在 "分析" 菜单中, 选择 "分析粒子" 并记录绘制区域表面的 "总面积" 值。</li>
		</ol>
		</li>
		<li>在电子表格中引入 PREGAP 和 POSTGAP 的总面积值, 以量化每个样本集的绝对迁移, 作为间隙表面测量的差异: PREGAP − POSTGAP [任意单位]。另外, 将每个条件的绝对迁移数据规范化为控制样本: (采样/控制) * 100 [%]。 注意: 对于单独迁移和填充间隙的单元格, 独立于相干锋的形成, (例如, MDA-MB-231 的乳腺癌人细胞), 绝对迁移可以通过计数细胞侵入中心地带, 无论是在控制或处理过的样品。</li>
		<li>绘制量化输出 (请参见图 1)。必要时进行统计分析。</li></ol></li></ol>当比较两个条件或方差检验的分析时, 考虑学生的 t 检验, 以得到更多的条件。
	
	

2. 地形研究的人工迁移前沿试验
<ol>
	<li>细胞单层制备
	<ol>
		<li>在无菌条件下工作, 将一层消毒的圆形片放在空的培养基中, 直到盘子表面完全覆盖。 注: 最多12和33片适合5厘米和10厘米直径的板材, 分别。</li>
		<li>在培养基上, 在2或3天后, 轻轻地将足够数量的细胞聚集在一起, 使100% 汇合。确保没有片重叠相邻的片和防止片的浮动, 应用温和的压力与无菌微提示。每24-48 小时刷新介质。 注意: 每一个细胞线必须进行彻底的检测, 以确定细胞浓度和时间需要达到完全70-100。通常为 Mv1Lu 或 HaCaT 单元格, 分别为 2-3 x 106或 2.5-4 x 106 cells/10 cm 直径板。对于较小的板材, 必须缩小单元格数。</li>
		<li>细胞到达汇合处后, 取出血清补充培养基, 用新鲜无血清培养基代替。在进行人工伤口前, 将细胞保持在无血清培养基中24小时。 注: Mv1Lu 或 HaCaT 细胞没有特殊的承印物要求;然而, 对于易在无血清条件下自发分离的细胞系, 使用聚 l-赖氨酸溶液预的培养表面应考虑为10。</li>
	</ol>
	</li>
	<li>单层伤
	<ol>
		<li>使用无菌镊子, 轻轻地移动一个片到一个干净的10厘米板含有新鲜的无血清培养基。用镊子在外围片, 避免损坏单层。避免过度的压力, 可能导致片破损。</li>
		<li>通过在片中心的横线上拖动一条消毒的刀片来制造人为的伤口。拖3-4 毫米来回, 为了完全删除中央单层带。确保没有细胞残骸附着在受伤的边缘。 注: 在直径为12毫米的圆片上, 切口是在片的中部进行的。确保在主前侧留有足够大 (至少2毫米宽) 的细胞, 以避免片在以下步骤中倾斜。</li>
		<li>将受伤的片与面对向上的细胞转移到一个干净的6井板上, 其中含有至少2毫升新鲜无血清培养基。可容纳4受伤的片/井。</li>
		<li>用新鲜无血清培养基轻轻冲洗两次, 去除分离细胞。</li>
	</ol>
	</li>
	<li>实验程序和取样
	<ol>
		<li>用含有选定处理的新鲜培养基轻轻地在井中交换培养基。 注: 另外, 对于不干扰培养基同质性的化学品或化合物, 可以直接将治疗接种到培养基中。谨慎行事, 避免任何潜在的细胞脱离。</li>
		<li>保持板在细胞孵化器 (37 ° c, 5% CO2) 为期望的实验时间。</li>
		<li>通过固定细胞来阻止细胞迁移;在 PBS 中用1毫升4% 福尔马林轻轻地取代实验培养基。在 RT 处孵育15分钟。 注: 对于时间课程的实验, 从培养基中取出样品, 并将其固定在一个单独的盘子中, 以避免甲醛烟雾影响其他的, 正在进行的实验条件。</li>
		<li>清洗细胞去除过量福尔马林;用新的 PBS 将溶液轻轻地替换在井中。重复至少两次。 注: 在这个阶段, 在密封之后, 板材可以被存放在4° c 无限期地。</li>
	</ol>
	</li>
	<li>免疫荧光 (IF) 和拓扑分析
	<ol>
		<li>如在其他地方所描述的那样对个别片进行染色和成像3,4,11。<ol>
			<li>Permeabilize 在 PBS 的0.3% 海卫 X-100 溶液中浸泡片10分钟。</li>
			<li>使用以下阻断液块30分钟: 0.3% 牛血清白蛋白;10% 胎牛血清;5% 脱脂牛奶;0.3% 海卫 X-100 解决方案在 PBS。 注: 不含牛奶的堵塞液可提前准备并贮存在-20 ° c。</li>
			<li>在潮湿的室内, 在室温下孵育1小时, 在无牛奶的阻断溶液中稀释初级抗体。将片倒置在15µL 抗体溶液中, 以确保适当的分布。 注意: 抗体工作稀释是可变的, 将取决于使用的抗体。例如, 兔 anti-c-六月抗体需要1/100 的稀释。</li>
			<li>洗涤三次, 蘸片在0.1% 海卫 X-100 解决方案在 PBS。</li>
			<li>在无奶阻塞缓冲液中稀释的二次抗体中孵育片30分钟。 注意: 抗体工作稀释是可变的, 将取决于使用的抗体。例如, 山羊抗兔 (488) 需要1/400 稀释以获得最佳的分辨率。其他标记试剂, 如笔和 Hoechst-33258 可以添加到孵化缓冲在这个阶段。</li>
			<li>洗涤三次, 蘸片在0.1% 海卫 X-100 解决方案在 PBS。</li>
			<li>将片倒置在10µL 安装介质放置 (例如, Vectashield) 放置在显微镜幻灯片上。在夜间将幻灯片留在室温下, 以使安装介质硬化。之后, 在黑暗中无限期地存放在4° c。</li>
		</ol>
		</li>
		<li>获得荧光或共聚焦显微镜图像的结构变化发生在迁移前边缘。 注意: 拓扑研究可以通过平铺图片从迁移前到内单层。通过基于软件的局部荧光强度分析, 可以进行半定量研究。</li>
	</ol>
	</li>
</ol>

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M., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Migration: Developmental Methods and Protocols. , 97-110 (2011).</li><li>Nyegaard, S., Christensen, B., Rasmussen, 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=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Szybalski, W., Iyer, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+DNA+by+Enzymatically+or+Chemically+Activated+Mitomycins+and+Porfiromycins,+Bifunctionally+"Alkylating"+Antibiotics.">Crosslinking of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally "Alkylating" Antibiotics.</a> Fed Proc. 23, 946-957 (1964).</li><li>Tomasz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitomycin+C:+small,+fast+and+deadly+(but+very+selective).">Mitomycin C: small, fast and deadly (but very selective).</a> Chem Biol. 2 (9), 575-579 (1995).</li><li>Sherry, D. M., Parks, E. E., Bullen, E. C., Updike, D. L., Howard, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+using+silicone+elastomer+masks+for+quantitative+analysis+of+cell+migration+without+cellular+damage+or+substrate+disruption.">A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption.</a> Cell Adh Migr. 7 (6), 469-475 (2013).</li><li>Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+phospholipase+in+generating+lipid+second+messengers+in+signal+transduction.">Role of phospholipase in generating lipid second messengers in signal transduction.</a> FASEB J. 5 (7), 2068-2077 (1991).</li><li>Lampugnani, M. 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作者声明没有利益冲突。

在皮肤或粘膜的破坏, 屏障功能恢复的行动, 许多细胞类型, 包括成纤维细胞或上皮和免疫细胞。共同, 这些细胞经历了一个复杂的过程, 涉及细胞凋亡, 增殖, 分化, 重要的是, 成纤维细胞和上皮组织迁移, 这是最终的机制, 负责修复被破坏的组织和浅上皮间隙闭合1,12因此, 研究细胞迁移有助于精确描述上皮细胞的生理行为, 同时也确定伤口治疗的调节电位?...

细胞迁移是需要的伤口愈合, 因为它是负责最后关闭的上皮间隙和恢复破坏表面1。在动物模型中执行人工伤口允许在近生理条件下复制这个复杂的过程2。然而, 这种方法往往导致昂贵和复杂的实验程序, 可能缺乏精确的研究不同的过程, 由于复杂的性质, 伤口愈合过程。
体外上皮细胞系的培养为研究这些细胞在创伤愈合中所起的作用以及治疗对细胞迁移行为的影响提供了一种有益的替代动物模型。利用 non-confluent 培养的分子技术, 经常研究上皮细胞的生理特性3,4,5,6;然而, 上皮完整性的破坏通常是通过细微的机械切口来实现的。在细胞培养中, 这意味着可忽略的细胞数可能暴露在伤口间隙, 它们代表了一个太小的样本分子生物学技术。然而, 这些病变可以在显微镜下进行研究, 利用一些上皮细胞系的固有迁移特性, 如水貂肺上皮细胞 (Mv1Lu) 或自发永生化的人角质形成细胞 (HaCaT)。线.
在这里, 我们描述了一种显微镜方法, 它是适合获得定量数据的上皮细胞迁移在伤口愈合的情况下3,4,7,8。此外, 我们提出的其他方法, 有助于研究的定性分子和形态学的变化发生在上皮膜的迁移。总的来说, 这些方法提供了一个框架来研究的动力学和形态学的变化涉及上皮细胞的行为和反应的治疗过程中伤口愈合。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Biowest, Nuaill&amp;eacute;, France</td><td>L0102-500</td><td>Optional 10 % FBS supplement</td></tr><tr><td>Eagles&amp;rsquo;s Minimum Essential Medium (EMEM)</td><td>Lonza</td><td>BE12 -662F</td><td>Optional 10 % FBS supplement</td></tr><tr><td>L-Glutamine</td><td>Lonza</td><td>BE17-605E</td><td>Use at 2 mM</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Fisher Scientific, Waltham, MA USA</td><td>DE17603A</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>T4049</td><td>Dilute as appropriate</td></tr><tr><td>Poly-L-Lysine</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>P9155</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS) (10x)</td><td>Gibco by Life Technologies</td><td>14200-067</td><td>Dilute to 1x</td></tr><tr><td>24-well culture plates</td><td>BD FALCON//SARSTED</td><td>734-0020</td><td></td></tr><tr><td>6-well culture plates</td><td>SARSTEDT</td><td>83-3920</td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>E9644</td><td>Used 10 ng/mL</td></tr><tr><td>Round cover glass</td><td>MENZEL-GL&amp;Auml;SER</td><td>MENZCB00120RA020</td><td>SHORT DEPTH OF FIELD</td></tr><tr><td>Reinforced razor blade no. 743</td><td>Martor (through VWR)</td><td>MARO743.50</td><td></td></tr><tr><td>200 &amp;micro;l sterile aerosol pipet tips</td><td>VWR</td><td>732-0541</td><td></td></tr><tr><td>20 &amp;micro;l sterile aerosol pipet tips</td><td>VWR</td><td>732-0528</td><td></td></tr><tr><td>Digital camera coupled phase contrast microscope</td><td>Motic Spain</td><td>Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31</td><td></td></tr><tr><td>Confocal microscope</td><td>ZEISS Microimaging, Germany</td><td>LSM 510 META</td><td></td></tr><tr><td>10 cm Culture dish</td><td>BD FALCON</td><td>353003</td><td></td></tr><tr><td>Rabbit polyclonal anti c-Jun antibody</td><td>Santa Cruz Biotechnology</td><td>sc-1694</td><td>Used 1:100</td></tr><tr><td>Anti-rabbit IgG (polyclonal goat ) AF 488</td><td>Invitrogen</td><td>A11008</td><td>Used 1:400</td></tr><tr><td>Hoechst-33258</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>14530</td><td>Used 1:1000</td></tr><tr><td>Alexa Fluor 594 phalloidin (in methanol) (red)</td><td>Invitrogen</td><td>A12381</td><td>Used 1:100</td></tr><tr><td>Bovine Serum Albumin</td><td>Santa Cruz Biotechnology</td><td>SC-2323</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>T9284</td><td></td></tr><tr><td>Skim milk</td><td>BD DIFCO</td><td>232100</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health, USA</td><td>Release 1.50i</td><td></td></tr><tr><td>Zen LSM 510 image processing software</td><td>ZEISS Microimaging, Germany</td><td>Release 5.0 SP 1.1</td><td></td></tr></tbody></table>

microscopy based methods for the assessment of epithelial cell migration during in vitro wound healing

细胞迁移是伤口愈合的一个必需的方面。在研究动物模型上制造人工伤口往往导致昂贵和复杂的实验程序, 但可能缺乏精确性。体外上皮细胞系的培养为研究创面愈合过程中细胞迁移行为和治疗对这些细胞的影响提供了一个合适的平台。上皮细胞的生理学在 non-confluent 条件下经常被研究;然而, 这种方法可能不像自然伤口愈合的条件。通过机械手段破坏上皮细胞的完整性产生了一个现实的模型, 但可能会阻碍分子技术的应用。因此, 显微技术是最理想的研究上皮细胞迁移的体外.在这里, 我们详细介绍了两种具体的方法, 人工伤刮法和人工迁移前分析法, 可以分别获得定量和定性的数据, 对上皮细胞的迁移性能。

定量研究的人工伤口划痕法: 评估表皮生长因子 (EGF) 促进迁移:
EGF 是一个众所周知的诱导上皮细胞增殖和迁移, 从而对量化迁移促进的积极控制。Mv1Lu 和 HaCaT 细胞单分子膜用于创面划伤的检测, 并获得了预处理图像。在接种 10 ng/毫升 EGF 后, 细胞孵育19小时前固定和后处理图片。通过维持血清饥饿状况, 使其增殖保持在最低限度。?...

Watch this Scientific Journal Video about Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing at JoVE.com

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

本手稿描述了如何在培养的上皮细胞单分子膜切口样病变, 方便模型伤口愈合在体外, 允许成像的共焦或激光扫描显微镜, 并能提供高质量定量和定性数据, 用于研究细胞行为和参与迁移的机制。

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 ...

microscopy-based-methods-for-assessment-epithelial-cell-migration

Research

JoVE Journal

Medicine

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

9.2K 次观看.  IMIB-Arrixaca. 本手稿描述了如何在培养的上皮细胞单分子膜切口样病变, 方便模型伤口愈合在体外, 允许成像的共焦或激光扫描显微镜, 并能提供高质量定量和定性数据, 用于研究细胞行为和参与迁移的机制。

视频: Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

科研

癌症研究

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 ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

生物工程

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

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

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

生物学

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 ...

医学

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.