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)。
1天
<ol><li>
细胞播种
	<ol>
<li>种子 2 106 SQ20B 细胞在12毫升培养基中 (cm) 在175厘米2烧瓶中72小时前一天, 允许细胞生长到80% 细胞汇合。<ol>
<li>为 SQ20B 细胞准备 CM, 补充 Dulbecco 的改良鹰培养基 (DMEM) 与10% 胎小牛血清 (FCS), 0.04 毫克/毫升氢化可的松, 100 U/毫升青霉素, 和0.1 克/L 链霉素。</li>
		</ol>
</li>
		<li>在层流罩下, 处理细胞。取出培养基, 用无菌磷酸缓冲盐 (PBS) 冲洗细胞, 并加入2.5 毫升的胰蛋白酶乙酸酸 (EDTA) (0.5 克/升)。孵育细胞5分钟在37° c, 然后停止反应, 增加12.5 毫升的 CM 温暖到37° c。使用单元格计数器计数单元格数。</li>
		<li>在 25 cm2烧瓶中的单元格中, 对每个要计算的条件的单元密度为 6 105单元格进行种子。在3毫升的 CM 中孵育24小时的细胞。</li>
	</ol>
</li>
</ol>2天
<ol start="2"><li>
细胞饥饿与包衣室的制备
	<ol>
<li>用0.1% 牛血清白蛋白 (BSA) 代替 FBS, 用3毫升的 low-fetal 牛血清 (FBS) 培养基取代每个烧瓶中的培养基, 使细胞挨饿。在孵卵器中将细胞饿死24小时。<ol>
<li>准备饥饿 cm (s cm), 补充 DMEM 与 0.1% BSA, 0.04 毫克/毫升氢化可的松, 100 U/毫升青霉素和0.1 克/L 链霉素。</li>
		</ol>
</li>
		<li>对于迁移化验, 请转到步骤1.3。</li>
		<li>为入侵试验, 12 小时在3天之前, 准备涂层的博伊登室通过增加500μ l CM 对每个被涂的房间。使用商业准备的涂层博伊登室, 并保持在4° c 使用前。将每个博伊登室放在伴板中, 并在37° c 过夜将其放在孵化器中。</li>
	</ol>
</li>
</ol>3天
<ol start="3"><li>
博伊登室的细胞播种
	<ol>
<li>使用伴板来制备趋化因子。用 10% FBS、0.04 毫克/毫升的氢化可的松、100毫升青霉素和0.1 克/l 链霉素, 填充24井伴侣板的750μ l 的完整培养基。<ol>
<li>适应每个细胞线和每个治疗条件的趋化因子。制备趋化蛋白 cm (ca cm), 补充 DMEM 与 10% FCS, 0.4 毫克/毫升的可的松, 100 U/毫升青霉素, 0.1 克/L 链霉素。</li>
		</ol>
</li>
		<li>将上部腔室转换为预伴板, 注意避免气泡。</li>
		<li>为入侵化验, 小心地从每个博伊登室去除450μ l CM。</li>
		<li>在层流罩下, 处理细胞。取出培养基, 用无菌 PBS 清洗细胞, 加入0.5 毫升的胰蛋白酶 (0.5 克/升), 然后在37° c 下孵育5分钟的细胞。通过增加 CM 加热到37° c 停止反应。使用单元格计数器计数单元格数。</li>
		<li>种子 3 104 SQ20B 细胞在500μ l 0.1% BSA 培养基中, 最后稀释 6 104细胞/毫升。 注意: 细胞浓度应适应每个细胞系。</li>
		<li>将板放入孵化箱中, 在37° c 为24小时。</li>
	</ol>
</li>
</ol>4天
<ol start="4"><li>
细胞固定和染色
	<ol>
<li>在特定于该单元格线的加倍时间之前修复单元格。在24小时前修复 SQ20B 细胞。</li>
		<li>从辅助板上取下每个插入物, 用棉签小心地从上部腔中取出细胞。这是特别重要的是要删除所有的细胞位于上腔。</li>
		<li>使用所提供的染色套件分别修复和着色每个插入, 以获得可格伦瓦德姬姆萨着色9 。另外, 使用4% 甲醛在另一个同伴板块中进行30分钟的固定。</li>
		<li>将插入物放在一个空的24井伴侣板下, 在实验室罩下晾干每个腔室。</li>
	</ol>
</li>
</ol>5天
<ol start="5"><li>
显微分析
	<ol>
<li>在20相衬显微镜上插入伴板与插入物, 并计算膜下部的每个迁移细胞。通过将目标从底部移到顶部来优化右焦点位置, 并停止膜底部的位置。</li>
		<li>计算迁移细胞数与种子细胞的比值。重复每个治疗条件的每三个计数。</li>
	</ol>
</li>
</ol>2. 刮伤试验: 细胞迁移
注意: 指令必须适应每种类型的单元格。
1天
<ol><li>
细胞播种
	<ol>
<li>种子 2 x 106 SQ20B 细胞在12毫升厘米在175厘米2烧瓶72小时前一天, 并允许细胞生长到80% 细胞汇合。</li>
		<li>在层流罩下, 处理细胞。取出培养基, 用无菌 PBS 洗涤细胞, 加入2.5 毫升的胰蛋白酶 EDTA (0.5 克/升), 并在37° c 下孵育5分钟的细胞。停止反应, 增加12.5 毫升的 CM 加热到37° c。使用单元格计数器计数单元格数。</li>
		<li>在 4 x 105/mL 的单元格密度下生成一个小修样本, 在每个井中给出每10μ l 的 4 4100单元格的最终密度。此浓度必须适应每个单元格线, 并且应在 1 x 104到 6 104单元格的范围内。</li>
		<li>通过沉积100μ l 在步骤2.1.2 中制备的溶液, 将种子细胞放入96井板的每一个井中。</li>
		<li>将板材置于室温下5分钟, 将细胞均匀地分散在井底。</li>
		<li>将板放入孵化箱中, 并允许电池在37° c 时附着在12至 16 h (最大) 的板上。</li>
	</ol>
</li>
</ol>2天
<ol start="2"><li>
刮伤试验
	<ol>
<li>从孵化器中取出步骤2.1 中准备的盘子。</li>
		<li>在引擎盖下, 根据制造商的协议, 用商业伤人装置制造伤口。</li>
		<li>立即从每个井中取出介质, 使用一个适应锥尖端的吸管, 注意不要碰到伤口。</li>
		<li>两次洗涤细胞通过重复的愿望和使用100μ l CM 温暖到37° c 每井。</li>
		<li>在洗涤步骤后, 用吸管与一个适应的锥形尖端移除 CM。</li>
		<li>每一个良好的治疗条件, 添加100μ l 介质。</li>
		<li>尽量避免在油井中产生气泡。反向移技术在这里可能会有所帮助。或者, 用注射器针除去气泡。</li>
		<li>把盘子放到视频显微镜的改装架上。</li>
		<li>为了提高图像质量, 允许板在第一次扫描前至少预热15分钟, 以避免板的下侧凝结。</li>
		<li>计划扫描的时间表, 使用视频显微镜软件在一个图像每井。入侵迁移试验需要最大2小时间隔。如果实验的目的是制作一个视频, 一个间隔30分钟的最大值是首选。</li>
		<li>在实验结束时, 仔细清洗伤人装置, 并按照制造商指示的四洗涤步骤进行操作。</li>
	</ol>
</li>
</ol>天 3, 4, 和5
<ol start="3"><li>
利用视频显微镜进行偏移分析
	<ol>
<li>至少24小时和5天, 监测和检查细胞迁移。</li>
		<li>使用适合每个单元格类型的适当的单元掩码来分析单元格迁移。若要获取适合于每个单元格线的单元掩码, 请使用特定的单元格图像集合从软件生成单元格处理定义。</li>
		<li>跟踪曲线并将数据导出到电子表格, 可用于分析和比较结果。</li>
	</ol>
</li>
</ol>3. 刮伤试验: 细胞侵袭
注意: 指令必须适应每种类型的单元格。
1天
<ol><li>
细胞播种
	<ol>
<li>如步骤2.1 所述, 对单元播种使用相同的协议。</li>
	</ol>
</li>
</ol>2天
<ol start="2"><li>
准备 ECM
	<ol>
<li>在4° c 使用前, 将基体解冻至少12小时。确保没有可见的聚合。如果聚集体是可见的, 保持矩阵在4° c 较长的时期, 直到聚合体消失。把母体放在冰上使用冷吸管提示。 注: 如果不保持在4° c, 基体将凝固。</li>
		<li>在冰上冷却离心管5分钟。</li>
		<li>采取 CM 冷却到4° c 从冰箱和稀释基体在冷离心管子获得最后的集中300微克/毫升。</li>
		<li>返回离心管与小修矩阵到冰箱在4° c。 注: 适用于离心管的冷却机架有助于在整个实验中保持4° c 的管子。</li>
	</ol>
</li>
</ol>2天
<ol start="3"><li>
ECM 的伤伤分析及处理
	<ol>
<li>从孵化器中取出步骤3.1 中准备的盘子。</li>
		<li>在引擎盖下, 根据制造商的协议使伤口使用商业伤人装置。</li>
		<li>立即从每个井中取出介质, 使用吸管与适应的锥形尖端, 注意不要触摸伤口。</li>
		<li>通过重复吸入程序和使用100μ l 的 CM 加热到37° c, 清洗两次细胞。</li>
		<li>第二次洗涤后, 将板放在4° c 恒温箱上, 平衡其温度为5分钟。</li>
		<li>取出每个井中的冷媒, 用锥形尖端吸管, 小心地从边缘移开吸管, 避免碰到伤口。</li>
		<li>在4° c 的情况下使用冷锥尖, 将50μ l 的小修矩阵添加到每个井中。</li>
		<li>把盘子放在37° c 的孵卵器里30分钟。 注:37 ° c 的 prewarmed 支撑架有助于加速96井板的升温。</li>
		<li>从孵化箱中取出盘子, 并在每种治疗条件下加100μ l 的适应 CM。</li>
		<li>尽量避免在油井中产生气泡。反向移技术在这里可能会有所帮助。或者, 用注射器针除去气泡。</li>
		<li>在视频显微镜下把盘子放到适应的货架上。</li>
		<li>为了提高图像质量, 允许板在第一次扫描前至少预热15分钟, 以避免板的下侧凝结。</li>
		<li>计划扫描的时间表, 使用视频显微镜软件在一个图像每井, 在划伤扫描模式。入侵迁移试验所需的最大2小时间隔。如果实验的目的是制作一个视频, 一个间隔30分钟的最大值是首选。</li>
		<li>在实验结束时, 仔细清洗伤人装置, 并按照制造商指示的四洗涤步骤进行操作。</li>
	</ol>
</li>
</ol>天 3, 4, 和5
<ol start="4"><li>
利用视频显微镜对细胞浸润进行分析。
	<ol>
<li>重复步骤2.3。</li>
	</ol>
</li>
</ol>

<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>

作者没有什么可透露的。

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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	<tbody>
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			<td height="21" style="width:216px;height:21px;">Fetal Calf Serum Gold</td>
			<td style="width:117px;">GE Healthcare</td>
			<td style="width:181px;">A15-151</td>
			<td style="width:339px;"></td>
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		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Hydrocortisone water soluble</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:181px;">H0396-100MG</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Penicillin/Streptomycin 100 X</td>
			<td style="width:117px;">Dominique Dutscher</td>
			<td style="width:181px;">L0022-100</td>
			<td style="width:339px;"></td>
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		<tr height="21">
			<td height="21" style="width:216px;height:21px;">DMEM</td>
			<td style="width:117px;">Gibco</td>
			<td style="width:181px;">61965-026</td>
			<td style="width:339px;"></td>
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		<tr height="21">
			<td height="21" style="width:216px;height:21px;">F12 Nut Mix (1X) + GlutaMAX-I</td>
			<td style="width:117px;">Gibco</td>
			<td style="width:181px;">31765-027</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">EGF</td>
			<td style="width:117px;">Promega</td>
			<td style="width:181px;">G5021</td>
			<td style="width:339px;">The solution must be prepared just before use because it is very unstable</td>
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		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Z1 coulter particle</td>
			<td style="width:117px;">Beckman Coulter</td>
			<td style="width:181px;">6605698</td>
			<td style="width:339px;"></td>
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		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Optical microscope</td>
			<td style="width:117px;">Olympus&#160;</td>
			<td style="width:181px;">CKX31</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="63">
			<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>
			<td style="width:181px;">-</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Wound Maker</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">4494</td>
			<td style="width:339px;">Store in safe and dry place</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">96-well ImageLock Plate</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">4379</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">CoolBox 96F System with CoolSink 96F</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">1500-0078-A00</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">CoolBox with M30 System</td>
			<td style="width:117px;">Essen Bioscience</td>
			<td style="width:181px;">1500-0078-A00</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Boyden Insert</td>
			<td style="width:117px;">Dominique Dutcher</td>
			<td style="width:181px;">353097</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Boyden Coated Insert</td>
			<td style="width:117px;">Dominique Dutcher</td>
			<td style="width:181px;">354483</td>
			<td style="width:339px;">Store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">BD Matrigel standard</td>
			<td style="width:117px;">BD BioScience</td>
			<td style="width:181px;">BD 354234</td>
			<td style="width:339px;">Store at -20&#176;C.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">RAL 555 Staining Kit</td>
			<td style="width:117px;">RAL Diagnostics</td>
			<td style="width:181px;">&#160;361550</td>
			<td style="width:339px;">Store in safe and dry place</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Microcentrifuge tubes</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:181px;">33511</td>
			<td style="width:339px;"></td>
		</tr>
	</tbody>
</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。

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

evaluation-cell-invasion-migration-process-comparison-video

Research

JoVE Journal

Cancer Research

17.5K 次观看.  Université Lyon 1. 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。

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

用点法分析细胞板的迁移

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

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. ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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 ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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 ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...