Name: evaluation of the cell invasion and migration process: a comparison of the video microscope-based scratch wound assay and the boyden chamber assay
Uploaded: 2017-11-17T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay at JoVE.com

这些技术是在 LabEx 素数 (ANR-11-LABX-0063) 的支持下开发的, 合同计划-地区 (CPER) 在星辰广场 (CPER 2009-2013) 的科学框架内, 以及抒情格补助金 INCa-DGOS-4664。

注: 不含 ecm 的博伊登室和划痕分析被称为迁移试验, 与 ecm 相同的化验被称为入侵检测。
1. 博伊登室试验
注: 本协议适用于 SQ20B 细胞系, 这是从复发头颈部鳞状细胞癌 (HNSCC) 喉癌, 并获得从约翰小 (波士顿, 马萨诸塞州, 美国)。
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细胞播种
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<li>种子 2 106 SQ20B 细胞在12毫升培养基中 (cm) 在175...

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Moncharmont, 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=Radiation-enhanced+cell+migration/invasion+process:+A+review.">Radiation-enhanced cell migration/invasion process: A review.</a> Crit Rev Oncol Hematol. , (2014).</li><li>Burdsal, C. A., Damsky, C. H., Pedersen, R. 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+role+of+E-cadherin+and+integrins+in+mesoderm+differentiation+and+migration+at+the+mammalian+primitive+streak.">The role of E-cadherin and integrins in mesoderm differentiation and migration at the mammalian primitive streak.</a> Development. 118 (3), 829-844 (1993).</li><li>Chen, C., Zimmermann, M., Tinhofer, I., Kaufmann, A. M., Albers, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+and+cancer+stem(-like)+cells+in+head+and+neck+squamous+cell+carcinoma.">Epithelial-to-mesenchymal transition and cancer stem(-like) cells in head and neck squamous cell carcinoma.</a> Cancer Lett. 338 (1), 47-56 (2013).</li><li>Nelson, A. R., Fingleton, B., Rothenberg, M. L., Matrisian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases:+biologic+activity+and+clinical+implications.">Matrix metalloproteinases: biologic activity and clinical implications.</a> J Clin Oncol. 18 (5), 1135-1149 (2000).</li><li>Smith, A., Teknos, T. N., Pan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+to+mesenchymal+transition+in+head+and+neck+squamous+cell+carcinoma.">Epithelial to mesenchymal transition in head and neck squamous cell carcinoma.</a> Oral Oncol. 49 (4), 287-292 (2013).</li><li>Chen, H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Meth Mol Biol. 294, 15-22 (2005).</li><li>Rodriguez, L. G., Wu, X., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-healing+assay.">Wound-healing assay.</a> Meth Mol Biol. 294, 23-29 (2005).</li><li>Piaton, E., 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=Technical+recommendations+and+best+practice+guidelines+for+May-Gr&#252;nwald-Giemsa+staining:+literature+review+and+insights+from+the+quality+assurance.">Technical recommendations and best practice guidelines for May-Gr&#252;nwald-Giemsa staining: literature review and insights from the quality assurance.</a> Ann Path. 35 (4), 294-305 (2015).</li><li>Moncharmont, 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=Targeting+a+cornerstone+of+radiation+resistance:+cancer+stem+cell.">Targeting a cornerstone of radiation resistance: cancer stem cell.</a> Cancer lett. 322 (2), 139-147 (2012).</li><li>Moncharmont, 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=Carbon+ion+irradiation+withstands+cancer+stem+cells’+migration/invasion+process+in+Head+and+Neck+Squamous+Cell+Carcinoma+(HNSCC).">Carbon ion irradiation withstands cancer stem cells’ migration/invasion process in Head and Neck Squamous Cell Carcinoma (HNSCC).</a> Oncotarget. 7 (30), 47738-47749 (2016).</li><li>Gilormini, M., Wozny, A. -. S., Battiston-Montagne, P., Ardail, D., Alphonse, G., Rodriguez-Lafrasse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+a+Head+and+Neck+Squamous+Cell+Carcinoma+Subpopulation+Having+Stem+Cell+Characteristics.">Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics.</a> J Vis Exp: JoVE. (111), (2016).</li></ol>

我们在这里报告两种不同的模式来研究细胞入侵和迁移过程。对这一过程的分析对于了解转移复发的因素是很重要的, 这可能是由于癌细胞亚群的增加运动而解释为肿瘤干细胞10,11。
博伊登室实验是研究细胞侵袭和迁移的最常用技术之一。这种方法的一个优点是它的重现性, 虽然这项技术是高度依赖于实验者的专长。细胞计数是手动...

细胞侵袭和迁移参与癌细胞的传播, 这是抵抗治疗的主要原因1 , 并可导致癌症治疗后的局部或转移复发2。上皮-间充质过渡 (急诊) 是细胞浸润-移行的初始过程, 癌细胞从上皮细胞转变为间质表型。e-钙黏蛋白是上皮表型的胞外标记物3, 增加的 n-钙黏蛋白和波形的表达是骨髓间质表型的特征4。迁移还取决于癌细胞的内在能力, 通过矩阵蛋白酶5的作用侵入细胞外基质 (ECM)。
这种 invasion–migration 机制已经被描述为癌症在许多地方, 特别是头颈部癌症6。许多研究人员把重点放在迁移和入侵过程上, 以便更好地理解癌细胞的传播, 希望这些知识能导致新的治疗策略。这是至关重要的, 这些研究是使用可靠和重现性的化验。
体外对细胞运动的分析可能具有挑战性。多年前, 博伊登室检测被认为是 invasion–migration 分析的标准7。然而, 它是费时, 往往是不准确的。第二个测试是伤口愈合试验8, 它涉及在细胞单层培养上进行划痕, 并在固定时间间隔捕获细胞入侵和迁移的图像。由于连续两次试验的结果相差很大, 这一技术受到了广泛的批评。然而, 现代技术的应用, 特别是在显微镜下, 提高了刮伤试验的重现性。视频显微镜可以很容易地在孵化器中引入, 并能产生细胞迁移的 real-time 图像。这些装置记录了显微镜数据, 并提供了一段时间内缠绕细胞汇流的自动分析。本文的目的是描述博伊登室法和优化的刮伤法, 并讨论每种方法的优缺点。

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			<td height="42" style="width:216px;height:42px;">EGF</td>
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			<td style="width:339px;">The solution must be prepared just before use because it is very unstable</td>
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			<td style="width:181px;">CKX31</td>
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			<td height="63" style="width:216px;height:63px;">SQ20B cell line</td>
			<td style="width:117px;">Gift from the John Little&#8217;s Laboratory</td>
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			<td style="width:339px;">Store in safe and dry place</td>
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			<td style="width:181px;">1500-0078-A00</td>
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			<td height="42" style="width:216px;height:42px;">Boyden Insert</td>
			<td style="width:117px;">Dominique Dutcher</td>
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			<td style="width:339px;">Store at -20 &#176;C</td>
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			<td height="42" style="width:216px;height:42px;">Companion 24-well Plate</td>
			<td style="width:117px;">Dominique Dutcher</td>
			<td style="width:181px;">353504</td>
			<td style="width:339px;"></td>
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			<td height="21" style="width:216px;height:21px;">BD Matrigel standard</td>
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			<td height="21" style="width:216px;height:21px;">Microcentrifuge tubes</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:181px;">33511</td>
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</table>

evaluation of the cell invasion and migration process: a comparison of the video microscope-based scratch wound assay and the boyden chamber assay

肿瘤细胞的侵袭和迁移能力是癌症进展和复发的主要因素。许多研究探索了迁移和入侵的能力, 以了解癌症细胞传播, 目的是制定新的治疗策略。分析这些能力的细胞和分子基础, 导致细胞的移动性和理化性质的骨架和细胞微环境的表征。多年来, 博伊登室法和刮伤法是研究细胞侵袭和迁移的标准技术。然而, 这两种技术有局限性。博伊登室法检测困难, 费时, 刮伤法重现性低。现代技术的发展, 特别是在显微镜下, 增加了刮伤试验的重现性。利用强大的分析系统, "孵化器" 的视频显微镜可以提供自动和 real-time 的细胞迁移和入侵分析。本论文的目的是报告和比较两种方法用于研究细胞侵袭和迁移: 博伊登室法和优化的体外视频 microscope-based 刮伤法。

我们在这里报告两种不同的方法来分析细胞入侵和迁移。图 1显示了博伊登室实验。插入物被放置在具有趋化介质的伴板中, 细胞在 CM 中播种。该膜可以被涂覆 (迁移化验) 或涂层 (入侵检测)。细胞在 s CM 中被播种入上部房间。下部腔内填充 CM 作为趋化因子。细胞在加倍的时间之前被固定。
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Watch this Scientific Journal Video about Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay at JoVE.com

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

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

本研究报告了两种不同的细胞浸润和迁移分析方法: 博伊登室法和体外视频 microscope-based 伤口愈合试验。对这两种实验的协议进行了描述, 并对它们的优缺点进行了比较。

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