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>

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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。

<table><tbody><tr><td>Fetal Calf Serum Gold</td><td>GE Healthcare</td><td>A15-151</td><td></td></tr><tr><td>Hydrocortisone water soluble</td><td>Sigma-Aldrich</td><td>H0396-100MG</td><td></td></tr><tr><td>Penicillin/Streptomycin 100 X</td><td>Dominique Dutscher</td><td>L0022-100</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>61965-026</td><td></td></tr><tr><td>F12 Nut Mix (1X) + GlutaMAX-I</td><td>Gibco</td><td>31765-027</td><td></td></tr><tr><td>EGF</td><td>Promega</td><td>G5021</td><td>The solution must be prepared just before use because it is very unstable</td></tr><tr><td>Z1 coulter particle</td><td>Beckman Coulter</td><td>6605698</td><td></td></tr><tr><td>Optical microscope</td><td>Olympus&amp;nbsp;</td><td>CKX31</td><td></td></tr><tr><td>SQ20B cell line</td><td>Gift from the John Little&amp;rsquo;s Laboratory</td><td>-</td><td></td></tr><tr><td>Wound Maker</td><td>Essen Bioscience</td><td>4494</td><td>Store in safe and dry place</td></tr><tr><td>96-well ImageLock Plate</td><td>Essen Bioscience</td><td>4379</td><td></td></tr><tr><td>CoolBox 96F System with CoolSink 96F</td><td>Essen Bioscience</td><td>1500-0078-A00</td><td></td></tr><tr><td>CoolBox with M30 System</td><td>Essen Bioscience</td><td>1500-0078-A00</td><td></td></tr><tr><td>Boyden Insert</td><td>Dominique Dutcher</td><td>353097</td><td></td></tr><tr><td>Boyden Coated Insert</td><td>Dominique Dutcher</td><td>354483</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Companion 24-well Plate</td><td>Dominique Dutcher</td><td>353504</td><td></td></tr><tr><td>BD Matrigel standard</td><td>BD BioScience</td><td>BD 354234</td><td>Store at -20&amp;deg;C.&amp;nbsp;</td></tr><tr><td>RAL 555 Staining Kit</td><td>RAL Diagnostics</td><td>&amp;nbsp;361550</td><td>Store in safe and dry place</td></tr><tr><td>Microcentrifuge tubes</td><td>Eppendorf</td><td>33511</td><td></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

Universidad de Cartagena UDEC

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

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

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. ...

癌症研究

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

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 ...

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.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.