Name: a novel in vitro wound healing assay to evaluate cell migration
Uploaded: 2018-03-17T13:00:00.000+00:00
Duration: 8 min 55 s
Description: Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

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

1. 细胞制剂
<ol><li>种子 2.5x106细胞在10毫升的最低基本培养基 (内存) 补充2毫米谷氨酰胺 (MEMg), 加上10% 胎牛血清 (血清), 抗生素 (0.1 毫克/毫升青霉素和链霉素), 和嘌呤霉素 (0.5 µg/毫升的选择和维护细胞线) 在一个 T75 瓶。将烧瓶在37摄氏度和 5% CO2孵化2天。在开始实验之前, 检查细胞在倒置显微镜下的汇合。 注: 用于实验的细胞是永生化的人支气管上皮细胞转基因与慢病毒载体的载体, 赋予嘌呤霉素抗性。他们稳定地表达功能 CFTR14,15。</li>
	<li>一旦细胞的汇合达到 90-100%, 从烧瓶中吸取培养基, 并将其丢弃在生物安全柜 ii 类的废物瓶中。用6毫升磷酸盐缓冲盐水冲洗细胞, 不含钙和镁。手动摇动烧瓶并丢弃。 注意: 小心不要用吸管接触细胞单层。</li>
	<li>添加10毫升的37摄氏度和 5% CO2的烧瓶和孵化的瓶, 10 分钟。</li>
	<li>把它吸掉, 然后扔掉它。然后在烧瓶中加入2毫升的胰蛋白酶/EDTA。</li>
	<li>轻轻地摇动烧瓶, 允许溶液完全涂上细胞, 在37摄氏度和 5% CO2上孵化出的烧瓶10分钟, 直到细胞在显微镜下可见分离。 注: 在孵化时间结束时, 细胞应呈圆形, 不附着在塑料表面。如果细胞没有很好的分离, 人工搅拌可能是必要的。</li>
	<li>加入10毫升的 MEMg 加10% 的血清, 以灭活胰蛋白酶, 并通过洗涤烧瓶的底部收集细胞。将体积转换成锥形50毫升管。</li>
	<li>在 80 x g上离心管5分钟。</li>
	<li>在6毫升的 MEMg 加10% 的血清中, 吸入上清并重新悬浮细胞。用吸管反复分解任何团簇。</li>
	<li>用微取出10µL 细胞悬浮液, 并在先前放在 Burker 或 Neubauer 室的盖玻璃下注入体积。</li>
	<li>计算单元格数。</li>
</ol>2. 细胞播种在文化插入
<ol><li>在12井板的每一个井, 转移文化插入与无菌镊子 (图 1)。使用镊子按下插入边, 以便将它们固定在板材的表面。 注: 插入有一个粘性的底部, 允许粘合。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56825/56825fig1.jpg"> 图 1: 硅树脂培养基的示意图表示, 正确放入12井板的井中.<a href="//cloudflare.jove.com/files/ftp_upload/56825/56825fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>适当稀释 MEMg 中的细胞悬浮液加10% 的血清。用70µL 细胞悬架 (大约 3.5x104细胞/室) 填充插入物的每个隔间。 注意: 每个隔间中应用的细胞密度取决于细胞的类型。建议使用一个细胞密度, 导致24小时内完全汇合。</li>
	<li>在显微镜下, 检查细胞是否从插入隔间漏出, 并在37摄氏度和 5% CO2上孵化12井板24小时。</li>
</ol>3. 伪创伤愈合试验
<ol><li>孵化后, 在倒置显微镜下对细胞进行可视化, 以验证是否形成了汇合细胞膜。</li>
	<li>在1毫升的 MEMg 中重新挂起测试化合物 (例如,安培)。 注: 准备新鲜安培稀释从库存解决方案存储在-20 °c。</li>
	<li>用无菌镊子轻轻取出刀片。小心不要打破细胞膜。将插入物转移到吸水性纸上。 注: 要重新使用相同的刀片, 在70% 乙醇消毒, 至少3小时。建议以后把它们扔掉。</li>
	<li>要去除非黏附的细胞, 增加1毫升 MEMg 每井使用微。关上盘子, 轻轻地摇动它。 注: 请勿直接在细胞膜上添加培养基, 以避免其破裂。</li>
	<li>吸入培养基, 并用1毫升 MEMg。关闭板和可视化的无细胞间隙 (由插入创建) 下倒置显微镜下4X 放大, 配备了一个视频摄像头。获取图像的时间为零 (T0), 并将其保存为. jpg 格式。</li>
	<li>从水井中取出介质, 用1毫升的 PBS 冲洗, 然后丢弃。</li>
	<li>将测试化合物 (3.2 点准备) 添加到油井中。对于未经处理的对照样品, 添加1毫升的 MEMg。在37摄氏度和 5% CO2上孵化板材。</li>
	<li>15、20、24小时治疗后, 观察显微镜下的细胞迁移4X 倍放大, 获取图像。 注意: 在此步骤中, 尝试在与 T0 相同的区域中捕获图像。捕捉图像的时间间隔的选择取决于细胞迁移速度。</li>
	<li>为了研究某些选择性抑制剂的影响,即AG1478 在细胞迁移过程中, 在移除插入物之前从每个插入间隔中吸入介质。<ol>
<li>用新鲜的 MEMg 清洗每个车厢, 并用70µL MEMg 补充 AG1478。</li>
		<li>在37°c 和 5% CO2的孵化30分钟后, 按照从点3.3 的说明进行操作。 注: 在洗涤步骤和前处理的细胞膜与特定抑制剂, 小心不要删除插入。</li>
	</ol>
</li>
</ol>4. 图像分析
<ol><li>实验完成后, 选择各种实验组最具代表性的样本, 并在 T0、T15、T20 和 T24 h 上创建一个包含单个图像的 zip 文件。 注意: 在选定的时间间隔内为所有样本拍摄单个图像。运行 triplicates 为每个实验, 这是重复至少三次。最后, 对所有实验组, 分析了每一个时间点的至少3张图像 ("a"、"b"、"c"、等等, 分别从每个独立实验中推导出来)。</li>
	<li>将 zip 文件上载到图像分析软件中, 通过电子邮件自动提供电子表格摘要文件, 其中包含所选时间点上的单元格覆盖和划痕区域的实验数据 (百分比)。 注意: 对前缘和间隙区域的识别主要是基于边缘检测方法 (旨在识别图像亮度急剧变化的点)。</li>
	<li>保存数据并收集它们。在时间零处正常化所有数据的平均值。计算每个时间点上所有复制的规范化数据的平均值和相对标准误差 (SEM)。通过对方差进行双向分析, 用适当的统计软件进行统计分析。不同时间间隔的肽治疗组和对照组之间的差异被认为是对p< 0.05 的统计 signiﬁcant。</li>
	<li>将获得的数据绘制为一个直方图, 该图显示所有样本组的单元格覆盖区域的百分比 ( vs选定的时间间隔)。</li>
</ol>

<ol>
	<li>Enyedi, B., Niethammer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+epithelial+wound+detection.">Mechanisms of epithelial wound detection.</a> Trends Cell Biol. 25, 398-407 (2015).</li><li>Kujath, P., Kujath, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complicated+skin,+skin+structure+and+soft+tissue+infections+-+are+we+threatened+by+multi-resistant+pathogens?.">Complicated skin, skin structure and soft tissue infections - are we threatened by multi-resistant pathogens?.</a> Eur J Med Res. 15, 544-553 (2010).</li><li>Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulm Pharmacol Ther. 21, 595-599 (2008).</li><li>Chiappini, E., Taccetti, G., de Martino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lung+infections+in+cystic+fibrosis+patients:+an+update.">Bacterial lung infections in cystic fibrosis patients: an update.</a> Pediatr Infect Dis J. 33, 653-654 (2014).</li><li>Mangoni, M. L., McDermott, A. M., Zasloff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+and+wound+healing:+biological+and+therapeutic+considerations.">Antimicrobial peptides and wound healing: biological and therapeutic considerations.</a> Exp Dermatol. 25, 167-173 (2016).</li><li>Lau, K., Paus, R., Tiede, S., Day, P., Bayat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+role+of+stem+cells+in+cutaneous+wound+healing.">Exploring the role of stem cells in cutaneous wound healing.</a> Exp Dermatol. 18, 921-933 (2009).</li><li>Hu, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+and+regenerative+repair+in+wound+healing.">Tissue engineering and regenerative repair in wound healing.</a> Ann Biomed Eng. 42, 1494-1507 (2014).</li><li>Ramot, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+PPARgamma-mediated+signalling+in+skin+biology+and+pathology:+new+targets+and+opportunities+for+clinical+dermatology.">The role of PPARgamma-mediated signalling in skin biology and pathology: new targets and opportunities for clinical dermatology.</a> Exp Dermatol. 24, 245-251 (2015).</li><li>Lai, Y., Gallo, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPed+up+immunity:+how+antimicrobial+peptides+have+multiple+roles+in+immune+defense.">AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense.</a> Trends Immunol. 30, 131-141 (2009).</li><li>Zasloff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+of+multicellular+organisms.">Antimicrobial peptides of multicellular organisms.</a> Nature. 415, 389-395 (2002).</li><li>Hulkower, K. I., Herber, R. L. <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+and+invasion+assays+as+tools+for+drug+discovery.">Cell migration and invasion assays as tools for drug discovery.</a> Pharmaceutics. 3, 107-124 (2011).</li><li>Di Grazia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+frog+skin-derived+antimicrobial+peptide+esculentin-1a(1-21)NH2+promotes+the+migration+of+human+HaCaT+keratinocytes+in+an+EGF+receptor-dependent+manner:+a+novel+promoter+of+human+skin+wound+healing?.">The frog skin-derived antimicrobial peptide esculentin-1a(1-21)NH2 promotes the migration of human HaCaT keratinocytes in an EGF receptor-dependent manner: a novel promoter of human skin wound healing?.</a> PLoS One. 10, e0128663 (2015).</li><li>Di Grazia, A., 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=D-Amino+acids+incorporation+in+the+frog+skin-derived+peptide+esculentin-1a(1-21)NH2+is+beneficial+for+its+multiple+functions.">D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions.</a> Amino Acids. 47, 2505-2519 (2015).</li><li>Cappiello, F., 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=Esculentin-1a-derived+peptides+promote+clearance+of+Pseudomonas+aeruginosa+internalized+in+bronchial+cells+of+cystic+fibrosis+patients+and+lung+cell+migration:+biochemical+properties+and+a+plausible+mode+of+action.">Esculentin-1a-derived peptides promote clearance of Pseudomonas aeruginosa internalized in bronchial cells of cystic fibrosis patients and lung cell migration: biochemical properties and a plausible mode of action.</a> Antimicrob Agents Chemother. 60, 7252-7262 (2016).</li><li>Bebok, Z., 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=Failure+of+cAMP+agonists+to+activate+rescued+deltaF508+CFTR+in+CFBE41o-+airway+epithelial+monolayers.">Failure of cAMP agonists to activate rescued deltaF508 CFTR in CFBE41o- airway epithelial monolayers.</a> J Physiol. 569, 601-615 (2005).</li><li>Trinh, N. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+defective+cystic+fibrosis+airway+epithelial+wound+repair+after+CFTR+rescue.">Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue.</a> Eur Respir J. 40, 1390-1400 (2012).</li><li>Gan, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidermal+growth+factor+receptor+(EGFR)+tyrosine+kinase+inhibitor+AG1478+increases+the+formation+of+inactive+untethered+EGFR+dimers.+Implications+for+combination+therapy+with+monoclonal+antibody+806.">The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 increases the formation of inactive untethered EGFR dimers. Implications for combination therapy with monoclonal antibody 806.</a> J Biol Chem. 282, 2840-2850 (2007).</li><li>Tokumaru, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+keratinocyte+migration+via+transactivation+of+the+epidermal+growth+factor+receptor+by+the+antimicrobial+peptide+LL-37.">Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37.</a> J Immunol. 175, 4662-4668 (2005).</li><li>Tjabringa, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antimicrobial+peptide+LL-37+activates+innate+immunity+at+the+airway+epithelial+surface+by+transactivation+of+the+epidermal+growth+factor+receptor.">The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor.</a> J Immunol. 171, 6690-6696 (2003).</li><li>Di Grazia, A., Luca, V., Segev-Zarko, L. A., Shai, Y., Mangoni, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporins+A+and+B+stimulate+migration+of+HaCaT+keratinocytes+and+kill+intracellular+Staphylococcus+aureus.">Temporins A and B stimulate migration of HaCaT keratinocytes and kill intracellular Staphylococcus aureus.</a> Antimicrob Agents Chemother. 58, 2520-2527 (2014).</li></ol>

作者没有什么可透露的

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

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

<table><tbody><tr><td>Minimum essential medium (MEM)&amp;nbsp;</td><td>Euroclone</td><td>ECB2071L</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Glutamine</td><td>Euroclone</td><td>ECB3000D</td><td></td></tr><tr><td>Heat inactivated Fetal Bovine Serum (FBS)</td><td>Euroclone</td><td>ECS0180DH&amp;nbsp;</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Euroclone</td><td>ECB3001D</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>Trypsin/EDTA 1X in PBS</td><td>Euroclone</td><td>ECB3052D</td><td>Warm at room temperature before use</td></tr><tr><td>DPBS without calcium and magnesium (CMF-PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>DPBS with calcium and magnesium (PBS)</td><td>Sigma-Aldrich</td><td>D8662</td><td></td></tr><tr><td>Ibidi Culture-Insert 2 well</td><td>Ibidi&amp;nbsp;</td><td>80209</td><td></td></tr><tr><td>Wimasis Image Analysis</td><td>Ibidi&amp;nbsp;</td><td>30002</td><td></td></tr><tr><td>PRISM software&amp;nbsp;</td><td>GraphPad</td><td>version 6.0</td><td></td></tr></tbody></table>

a novel in vitro wound healing assay to evaluate cell migration

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

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

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

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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

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 ...

a-novel-in-vitro-wound-healing-assay-to-evaluate-cell-migration

Research

JoVE Journal

Immunology and Infection

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

21.7K 次观看.  Sapienza University of Rome. 在这里, 我们提出一个评估肽对支气管上皮细胞迁移的影响的协议。这种方法允许快速和高度重复获取的数量数据的速度的细胞迁移和伤口闭合。

视频: A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

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 ...

科研

发育生物学

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 ...

癌症研究

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 ...

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 ...

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

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 ...

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.