Name: in vitro cell migration and invasion assays
Uploaded: 2014-06-01T13:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about In vitro Cell Migration and Invasion Assays at JoVE.com

The authors thank Mike Myles, Sam Saunders, and C.W. Elton at the ECU Multimedia &amp; Technology Services for providing assistance in video production. We acknowledge the grant support from North Carolina Biotechnology Center, Golfers against Cancer, Brody Brothers Endowment Fund, American Heart Association, and ECU/Vidant Cancer Research and Education Fund (L.V.Y. and M.J.R.).

1，细胞培养伤口闭合含量<ol><li>使用0.25％胰蛋白酶-EDTA溶液中的组织培养板中分离细胞。沉淀的细胞在15毫升锥形管中，离心，吸出上清液，并重新悬浮的细胞在培养基中。铠细胞在6孔板的适当数量为在24小时内100％汇合。 注意：测试可能需要确定时间和细胞数达到100％汇合时，由于细胞的类型和该井所使用的大小（例如，1×10 6 B16F10黑素瘤细胞接种在一个良好的6孔板前24小时至伤口代）。可替换地，可以使用12孔或24孔板中。 注：如果可用，细胞可在处理过的细胞培养板使得伤口预铸被接种到文化 - 插入（从ibidi有限责任公司）。文化 - 插入可以通过枪头保护组织培养板的表面不受损坏。如果一个企业文化 - 插入时忽略步骤1.2。 </li><li>在无菌的环境（通常是生物安全罩）用200μl的移液管尖，以牢固地压在组织培养板的顶部，并通过细胞单层迅速地使一个垂直向下的伤口。不同尺寸的移液管尖端可以用来使所期望的伤口大小。不要过度使用武力的组织培养板吸头，因为它会损坏表面。如果伤口预铸，该步骤可以省略。 </li><li>小心吸媒体和细胞碎片。慢慢地对孔壁，以覆盖所述孔的底部，并避免额外的分离的细胞补充足够的培养基。以下伤口的产生和检测的初始图象，应采取。将组织培养板在培养箱中设定在适当的温度和CO 2浓度（通常为37℃和5％CO 2）。 </li><li>在几个时间点， 例如每3小时从incubat除去板或者，并将其放置在倒置显微镜下拍摄快照的图片，并检查伤口闭合。如果时间推移显微镜可用，拍照分钟的每隔X数量（通常以每5分钟为单位）持续时间在温度和CO 2控制腔。取决于细胞类型，伤口闭合的时间可能会发生变化。 </li><li>要分析的快照图片的结果，测量在伤口的一侧到另一使用比例尺的距离。用散点图或条形图， 例如 图1分析，并明确目前伤口闭合随着时间的推移。 </li><li>为了分析时间推移的图片，导入图片到ImageJ的软件（可自由查看的http://rsb.info.nih.gov/ij/）进行视频。要做到这一点打开ImageJ的，点击文件，导入，和图像序列。找到图片文件;单击该文件中的第一张照片。保存视频选择文件，另存为，和AVI。单个细胞的迁移可观察到恰拉cterize定向运动。此外，形态特征，如伪足形成和后缘回缩可以研究以及（ 图2和辅助视频）。 </li></ol> 2，Transwell小细胞迁移和入侵检测<ol><li>在无菌的环境（通常是生物安全罩）使用离心非酶细胞解离缓冲液，或0.25％胰蛋白酶-EDTA溶液中，沉淀的细胞分离的组织培养板中的细胞，并吸出的现有媒体留下沉淀的细胞。重悬的细胞在含有0.1％BSA（牛血清白蛋白）的无血清的细胞培养基。 注意：根据该细胞系被调查的0.25％胰蛋白酶-EDTA会影响细胞迁移和侵袭由于各种受体在细胞表面17的切割。 注意：每毫升的细胞数依赖于细胞的大小。进一步的测试，可能需要发现亲的接种密度志愿组织的最佳效果。通常为1×10 6个细胞/毫升是一个很好的起点，大多数细胞类型，当使用24孔Transwell插入。有一系列的transwell插入物的大小并增加细胞的数量应调整为相应。 </li><li>板100μl的细胞溶液在一个Transwell小插入过滤膜的顶部，并孵育10分钟，在37℃和5％CO 2，以使细胞沉淀下来。 注意：使用的transwell小室的膜的特定细孔径是依赖于细胞的样品中的各个尺寸。例如，如果该细胞是太小它们可以穿过的孔，而不需要促进迁移，或者如果它们太大，他们可能没有通过毛孔适合。有几个的transwell插入物与孔径大小的范围从3微米，5微米，8微米的用于细胞迁移测定。 Transwell小刀片是可商购自例如康宁公司。 注：Transwell小米igration分析可以很容易地修改，以执行细胞侵袭实验。要做到这一点，添加细胞外基质（ECM）材料上的Transwell小膜的顶部，然后对ECM的顶部添加细胞。例如，基质胶解冻和液化在冰上，然后30-50微升基质胶加入到24孔Transwell小插入和固化在37℃的培养箱中培养15-30分钟，以形成一个薄的凝胶层。细胞溶液在基质胶涂层的顶部加入，通过细胞外基质，以模拟的入侵。 注意：有Transwell小细胞迁移和Transwell小细胞侵袭实验之间有明显的差异。 Transwell小细胞迁移实验测定细胞走向化疗引诱的趋化能力。 Transwell小细胞侵袭测定法，但是，通过细胞外基质的同时测量细胞趋化性和侵袭的细胞，即通常在癌症转移或胚胎发育的发现过程。 </li><li>使用移液管，非常caref来自ully添加600微升所希望的化疗引诱到下部腔室在一个24孔板的底部。添加化学引诱而无需移动Transwell小插件，避免产生气泡。确保在底部的井的化疗引诱液，使接触带的膜在上部以及形成趋化梯度。温育时间是依赖于细胞类型和化学引诱被使用。 注：进一步的测试可能需要确定潜伏期。 注意：对于贴壁细胞，迁移的细胞将附着在膜1,8的另一侧。迁移的细胞进行定量可以执行以下步骤2.4到2.8（步骤2.4-2.8不需要在无菌环境中进行的）来执行。对于非粘附细胞，迁移的细胞将下降到介质中的下腔室。迁移细胞的数目可以通过使用血细胞计数器或算流式细胞仪5。 </li><li>从板块中删除Transwell小插件。使用棉签多次需要小心地取出，但没有从膜的顶部而不损坏它迁移媒体和剩余的细胞。 </li><li>加600-1,000微升70％乙醇到24孔板的孔中。将transwell小插件放入70％乙醇中10分钟，以使细胞固定。从24孔板取出Transwell小插入和使用棉签从膜的顶部取出剩余的乙醇。允许的transwell小膜干燥（通常10-15分钟）。 </li><li>加600-1,000微升0.2％的结晶紫为24孔板的孔和膜定位成它用于染色。在室温下孵育5-10分钟。 </li><li>轻轻地从膜的顶部用移液管尖端或棉棒取出结晶紫。非常小心，以避免洗去固定的细胞，可以根据需要沾膜进入蒸馏水多次以除去过量的晶体VIOLE万吨。允许的transwell小膜干燥。 </li><li>视图下方倒置显微镜和计数细胞在不同视场的数目，以得到通过膜迁移朝向化学引诱并附着在膜的下侧（ 图3）细胞的平均总和。 </li></ol>

<ol>
	<li>Castellone, R. D., Leffler, N. R., Dong, L., 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=Inhibition+of+tumor+cell+migration+and+metastasis+by+the+proton-sensing+GPR4+receptor.">Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor.</a> Cancer Lett. 312 (2), 197-208 (2011).</li><li>Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytoskeleton+and+cancer.">The cytoskeleton and cancer.</a> Cancer Metastasis Rev. 28, (2009).</li><li>Lauffenburger, D. A., Horwitz, A. F. <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:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>Peter, C., 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=Migration+to+apoptotic+&amp;#34;find-me&amp;#34;+signals+is+mediated+via+the+phagocyte+receptor+G2A.">Migration to apoptotic &amp;#34;find-me&amp;#34; signals is mediated via the phagocyte receptor G2A.</a> J. Biol. Chem. 283 (9), 5296-5305 (2008).</li><li>Radu, C. G., Yang, L. V., Riedinger, M., Au, M., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+chemotaxis+to+lysophosphatidylcholine+through+the+G2A+receptor.">T cell chemotaxis to lysophosphatidylcholine through the G2A receptor.</a> Proc. Natl. Acad. Sci. U. S. A. 101 (1), 245-250 (2004).</li><li>Buul, J. D., Hordijk, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+in+leukocyte+transendothelial+migration.">Signaling in leukocyte transendothelial migration.</a> Arterioscler Thromb. Vasc. Biol. (5), 824-833 (2004).</li><li>Yang, L. V., Heng, H. H., Wan, J., Southwood, C. M., Gow, A., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+promoters+and+polyadenylation+regulate+tissue-specific+expression+of+Hemogen+isoforms+during+hematopoiesis+and+spermatogenesis.">Alternative promoters and polyadenylation regulate tissue-specific expression of Hemogen isoforms during hematopoiesis and spermatogenesis.</a> 228 (4), 606-616 (2003).</li><li>Yang, L. V., Radu, C. G., Wang, L., Riedinger, M., Witte, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gi-independent+macrophage+chemotaxis+to+lysophosphatidylcholine+via+the+immunoregulatory+GPCR+G2A.">Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A.</a> Blood. 105 (3), 1127-1134 (2005).</li><li>Yoshida, M., Yoshida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chemotaxis+and+regulation+of+flagellar+movement+by+Ca2+.">Sperm chemotaxis and regulation of flagellar movement by Ca2+.</a> Mol. Hum. Reprod. 17 (8), 457-465 (2011).</li><li>Albini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+and+endothelial+cell+invasion+of+basement+membranes.+The+matrigel+chemoinvasion+assay+as+a+tool+for+dissecting+molecular+mechanisms.">Tumor and endothelial cell invasion of basement membranes. The matrigel chemoinvasion assay as a tool for dissecting molecular mechanisms.</a> Pathol. Oncol. Res. 4 (3), 230-241 (1998).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+determinants+of+metastasis.">Critical determinants of metastasis.</a> Semin. Cancer Biol. 12 (2), 89-96 (2002).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+mechanistic+insights+and+clinical+challenges.">Tumor metastasis: mechanistic insights and clinical challenges.</a> Nat. Med. 12 (8), 895-904 (2006).</li><li>Roussos, E. T., Condeelis, J. S., Patsialou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+in+cancer.">Chemotaxis in cancer.</a> Nat. Rev. Cancer. 11 (8), 573-587 (2011).</li><li>Zhang, 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=Comparative+study+of+3D+morphology+and+functions+on+genetically+engineered+mouse+melanoma+cells.">Comparative study of 3D morphology and functions on genetically engineered mouse melanoma cells.</a> Integr. Biol. (Camb. 4 (11), 1428-1436 (2012).</li><li>Junger, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purinergic+regulation+of+neutrophil+chemotaxis.">Purinergic regulation of neutrophil chemotaxis.</a> Cell. Mol. Life Sci. 65 (16), 2528-2540 (2008).</li><li>Lazennec, G., Richmond, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+chemokine+receptors:+new+insights+into+cancer-related+inflammation.">Chemokines and chemokine receptors: new insights into cancer-related inflammation.</a> Trends Mol. Med. 16 (3), 133-144 (2010).</li><li>Huang, H. L., 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=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J. Biomed. Sci. 17, (2010).</li><li>Cortesio, C. L., Boateng, L. R., Piazza, T. M., Bennin, D. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain-mediated+proteolysis+of+paxillin+negatively+regulates+focal+adhesion+dynamics+and+cell+migration.">Calpain-mediated proteolysis of paxillin negatively regulates focal adhesion dynamics and cell migration.</a> J. Biol. Chem. 286 (12), 9998-10006 (2011).</li><li>Wehrle-Haller, B., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+microtubules+and+focal+adhesion+dynamics+during+cell+migration.">Actin microtubules and focal adhesion dynamics during cell migration.</a> Int. J. Biochem. Cell Biol. 35 (1), 39-50 (2003).</li></ol>

The authors declare that they have no competing financial interests.

细胞迁移是在癌症研究，研究的一个重要方面，它也可以应用到发育，免疫和伤口愈合研究。细胞培养伤口闭合试验和Transwell小细胞迁移和侵袭测定法揭示了细胞迁移性的行为的详细信息，并且可以用于研究细胞迁移1,2,10,14的分子机制。我们的研究中使用这些细胞运动性测定法来确定B16F10黑色素瘤细胞系的迁移速度和侵袭能力。 细胞伤口愈合试验检查一个特定细胞系...

蠕动是活细胞的一个重要特征。细胞迁移是参与生命，胚胎发育，免疫反应，以及许多病理过程，如癌症转移和炎症1-9的概念。因此，方法来研究细胞的迁徙行为是非常有用的研究工具，在生物医学科学，生物学，生物工程及相关领域学科门类齐全的。 在癌症研究中的细胞迁移的研究是特别感兴趣，因为死亡的癌症患者的主要原因是关系到转移进程。为了让癌细胞扩散和传播遍及全身，癌细胞必须迁移和入侵，通过细胞外基质（ECM），intravasate进入血液循环，连接到一个远程站点，最后渗出，形成远处病灶1,10〜12。各种生物的方法可以用来详细研究这些事件。细胞培养伤口闭合的D中的Transwell小迁移和入侵检测被广泛应用于科学界1,10。这些测试可以提供必要的数据，可能会允许对如何以及特定细胞类型可以自发地迁移或响应一个化疗诱和定向朝向其迁移的理解。几个迁徙的表型进行了说明。细胞可以迁移见于间质或变形虫样运动或标记的集体迁移或细胞流13多细胞运动的单细胞形式如。在游动细胞用于运动的方法，可以使用细胞培养物的伤口闭合测定法可以容易地观察到。 在众多的方法来研究细胞迁移，细胞伤口缝合法是最简单的一种。这种方法可用于确定整个细胞团的迁移能力。时采取的一个步骤进一步它可以用于迁移14时，观察各个细胞的形态特征。随着伤口闭合的分析很多表型可能显露出来。比较控件时测量近距离随着时间的推移可能会发现特定迁移量的变化或受损迁徙的表型是前所未知的。此外，单细胞lamellipodium形成，尾部回缩，并定向运动可能会给什么可能会削弱或增强的利息14细胞的线索。 Transwell小迁移和侵袭测定法可用于分析单个细胞的定向到不同的化疗诱响应它们是否是趋化因子，生长因子，脂质，或核苷酸4,5,8,15,16的能力。它也可以评估差分迁移能力因过度表达的受体1,14的。这些测定法还可以用于鉴定和表征细胞迁移的关键调节剂，如Rho家族的小GTP酶2。下面的这些短且容易存取权限sible测试中，细胞迁移的模式和小区的侵入成三维基质的能力也可以被确定。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)</td><td>Gibco</td><td>11995-073</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A4503-50G</td><td></td></tr><tr><td>Trypsin EDTA 0.25%</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)</td><td>Gibco</td><td>14190-250</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma</td><td>C0775-100G</td><td>Dissolved in water at 0.2%</td></tr><tr><td>Cell disassociation buffer</td><td>Gibco</td><td>13151-014</td><td></td></tr><tr><td>Cell culture incubator</td><td>Thermo Fisher Scientific</td><td></td><td>Model # 3145</td></tr><tr><td>SterilGARD biosafety hood</td><td>The Baker Company, Inc.&amp;nbsp;</td><td></td><td>Model # VBM-600</td></tr><tr><td>EVOS Fl inverted microscope</td><td>Thermo Fisher Scientific</td><td></td><td>Model # AMF-4302-US</td></tr><tr><td>Tissue culture plate</td><td>Becton Dickinson</td><td>353046</td><td>Catalog number varies depending on the type of culture plate</td></tr><tr><td>Corning Transwell insert</td><td>Fisher</td><td>07-200-150</td><td>Catalog number varies depending on the pore size of the membrane</td></tr></tbody></table>

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.

这里提出的伤口闭合试验和Transwell小细胞迁移测定法使用小鼠B16F10黑素瘤细胞作为模型系统进行。在伤口愈合测定法，B16F10细胞接种在6孔组织培养板中并生长至24小时内100％汇合。使用使用时间推移显微镜记录枪头和伤口闭合（细胞迁移）生成约700μm宽的伤。可替换地，伤口闭合，也可以通过采取快照图片在不同时间点，如果时间推移显微镜不可用1研究。四张照片的时间推移系列在0，4,8...

Watch this Scientific Journal Video about In vitro Cell Migration and Invasion Assays at JoVE.com

In vitro Cell Migration and Invasion Assays

常用的，非常方便的方法研究细胞迁移和侵袭的体外描述。第一种方法是测量细胞运动的细胞伤口愈合测定法。第二种方法是，评估的趋化和细胞的侵袭能力Transwell小迁移和侵袭实验。

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 ...

in-vitro-cell-migration-and-invasion-assays

Universidad de Cartagena UDEC

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Cell Biology

Biology

In vitro Cell Migration and Invasion Assays

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

137.8K 次观看.  East Carolina University. 常用的，非常方便的方法研究细胞迁移和侵袭的体外描述。第一种方法是测量细胞运动的细胞伤口愈合测定法。第二种方法是，评估的趋化和细胞的侵袭能力Transwell小迁移和侵袭实验。 

视频: In vitro Cell Migration and Invasion Assays

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

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

小鼠乳腺肿瘤细胞在体外入侵测定中作为致癌细胞行为的测量

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

遗传学

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

医学

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

合成mRNA转染后肿瘤细胞迁移和侵袭的实时定量测量

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

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 ...