作者希望感谢 Bhagawat 萨勃拉曼尼亚, 伊 (杰森), 保罗 Randazzo 和卡罗尔的父母阅读和评论的手稿。这项工作得到了国立卫生研究院国家癌症研究所的校内研究项目的支持。

1. 盘子的涂层 (1 天)
注意: 在处理时, 请务必不要将指纹或污垢留在盘子底部。
<ol><li>解冻小鼠胶原 iv 在冰上, 和稀释它与50毫米 HCl (pH 1.3) 准备3毫升的10µg/毫升胶原蛋白 iv 溶液。 注: 胶原蛋白在37° c 时会沉淀。因此, 保持胶原蛋白溶液在低温下融化并与之一起工作是很重要的。避免重复的冻融循环, 准备适当大小的等分和存储在-80 ° c。</li>
	<li>添加250µL 胶原 IV 溶液, 每井的12井玻璃底板, 并把它们放在一个适当大小, 紧合塑料盒。将湿纸巾放在盘子周围, 制造一个潮湿的房间并关上盒子。在室温下一夜之间孵育盘子。 注: 由于只有生长区域需要涂敷, 所以只有覆盖的盖子滑动, 这是连接到板的底部, 与胶原蛋白溶液 (请参见图 1a, B的示意图板)。</li>
	<li>第二天早上, 冲洗盘子与去离子 H2O 两次去除不吸收的胶原蛋白和缓冲。将水直接放到板的边缘, 而不是由板底和片形成的边缘 (如果水被 pipetted 到这个区域就会飞溅)。空气干燥板在层流罩。立即用盘子或在4° c 贮存5天。 注: 不同的细胞系可能需要不同的涂层, 如纤维连接蛋白或胶原蛋白 i。</li>
</ol>2. 电镀点检测 (2 天)
<ol><li>
准备细胞悬浮
	<ol>
<li>要准备细胞悬浮, 使用在 DMEM/F12 培养基中生长的60毫米组织培养皿中的细胞, 辅以5% 匹马血清至80% 汇合</li>
		<li>用钙和镁 Dulbecco 的磷酸盐缓冲盐水 (DPBS) 冲洗细胞一次, 增加500µL 胰蛋白酶-EDTA (室温), 并孵育细胞为 3-5 分钟在一个孵化器在37° c, 5% CO2, 湿润的气氛。</li>
		<li>悬浮在4.5 毫升培养基中的分离细胞停止胰蛋白酶活性和计数20µL 分的细胞悬浮在例。</li>
		<li>将剩余的细胞在15毫升锥形管中通过离心 200 x g 进行3分钟的颗粒, 吸取上清液, 并重培养基中的细胞到 3 x 106细胞/毫升。 注: 对于其他涂层基底和/或细胞系, 最佳的细胞密度必须建立在稀释系列。建议将 3 x 106单元/mL 作为起始点。</li>
	</ol>
</li>
	<li>
电镀电池
	<ol>
<li>将10µL 的细胞悬浮液放在 IV 型胶原涂层12井玻璃底板的每个盖板的中心, 而不触及或刮伤涂层 (图 1a, B)。孵育板为30分钟, 在37° c, 湿润的气氛, 让细胞附着。在倒置显微镜中检查细胞的附着。 注意: 这在下落的边缘可以是最好看见的, 在那里单层的形成可以被观察 (图 1C)。如有必要, 可将钢板孵育至 3 h. 确保孵化箱内的湿度足够高, 因为水滴可以快速干燥。如果在这一步的下降量减少观察, 增加 PBS 的空间之间的井, 以增加湿度。</li>
		<li>用1毫升培养基轻轻冲洗两次, 去除非附着细胞。每井添加1毫升培养基。检查一个倒置的显微镜, 没有漂浮的细胞保持, 否则洗井再次。在37° c 的夜间孵育细胞, 5% CO2, 湿润的气氛, 获得良好定义的细胞表。 注: 漂浮细胞很可能会重新附着在盘子上, 形成不受欢迎的细胞群。</li>
	</ol>
</li>
</ol>3. 刺激细胞 (3 天)
<ol><li>使用反显微镜检查所有水井, 确保细胞菌落生长正常。如有必要, 标记不合适的水井, 不要使用。</li>
	<li>为所调查的每个条件适当地标记每一个板块。每一个条件运行一式两份或一式三份。</li>
	<li>改变培养基, 使其在细胞被刺激之前, 在含有0.1% 马血清 (饥饿培养基) 的 DMEM/F12 3 小时内使细胞挨饿。</li>
	<li>通过添加 EGF (5 ng/毫升最终浓度) 或其他所需的介质来刺激细胞, 用1毫升的 micropipettor 或旋转盘子轻轻地混合培养基。</li>
	<li>孵育该板块 1-4 天, 以观察边缘位移和边缘轮廓, 如第4.1 节所述, 或执行时间推移成像, 如在4.2 节所述。</li>
</ol>4. 细胞菌落和细胞迁移的可视化
<ol><li>
苏木精-曙红 (he) 染色测量菌落生长 (日 4-7)
	<ol>
<li>将70% 乙醇 (1 毫升/井) 的细胞固定在2分钟。</li>
		<li>染色核与苏木精 (1 毫升/井), 〜2分钟。</li>
		<li>用自来水冲洗细胞 (1 毫升/井) 直到细胞核是蓝色的, 5-10 分钟。</li>
		<li>染色细胞质与曙红 (1 毫升/井), 〜2分钟。</li>
		<li>用去离子水冲洗 (1 毫升/井)。 注意: 苏木精和红素溶液可以收集和使用多次, 直到染色变得微弱。</li>
		<li>空气干燥板。</li>
		<li>翻转板, 用尺子测量圆点的直径。或者, 用相机拍摄底片, 并分析 ImageJ 中的圆点直径。</li>
	</ol>
</li>
	<li>
延时显微镜 (3 天)
	<ol>
<li>打开孵化器显微镜, 将温度调整到37° c。用 dH 填充加湿器2o 将 CO2调整为5%。确保孵化室的湿化和机动阶段可以自由移动。关闭孵化室, 并允许显微镜调整到37° c。 注: 在这里使用的显微镜 (见材料表) 应加热到37° c, 至少3小时, 然后细胞被成像, 以避免漂移的焦点。</li>
		<li>把盘子放在孵化器显微镜的舞台上, 并设置舞台列表。图像两个对立的边缘和每个单元格点的中心 (图 1A)。</li>
		<li>使用10X 目标, 每3分钟拍摄一次图像, 15-24 小时。下载数据并在选择的程序中进行图像分析,例如、ImageJ 或 Matlab。</li>
	</ol>
</li>
</ol>

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作者没有什么要透露的。

在肿瘤进展过程中, 细胞从原发组织迁移到周围组织, 转移到远处的部位15。细胞群的迁移可以在点检测中观察到。在这里, 通过分析人乳腺癌细胞在 EGF 中的迁移表型来说明点测定。
为了获得准确和可复制的结果, 在点分析的设定中的几个步骤是至关重要的。首先, 板的涂层决定是否和如何强细胞能坚持板和如果细胞迁移。应注意的是, 该板的涂层与胶原 IV 或其他细胞外基质是均匀的, 以尽量减少实验的变异性。此外, 必须确保所研究的细胞在涂层板上附着和迁移, 并在多个浓度不同的基质中进行多次试验, 以优化所选细胞系的涂层条件。第二, 准确的细胞电镀是至关重要的重现性。细胞下降的足迹决定了细胞点的大小以及它的形状。理想情况下, 下落是放置在没有吸管尖端触及表面。下落的形状然后主要由板材表面的表面张力确定, 导致圆的足迹可再生直径。因此, 改变细胞悬架的体积可以改变细胞点的直径。
因此, 在执行点检测时, 需要考虑几个问题。首先, 细胞悬浮细胞的密度需要调整到涂层以及所使用的细胞类型。一般而言, 如果水滴扩散得更宽, 则需要更高的细胞密度, 才能将细胞板成汇合的单分子膜。此外, 更多的细胞需要被镀, 以获得一个汇合单层, 如果细胞是小的, 并没有扩散, 相比大, 传播细胞。其次, 点分析可以采用不同的板格式, 并为此, 网点大小可以适应较小的水井, 减少了点镀, 如果需要, 更大的点可以通过使用更高的下降量。第三, 有些细胞线不会在30分钟内附着在板上, 在这种情况下, 电镀时间可以延长到3小时。同样的, 涂层的板必须是合适的细胞迁移, 并注意不要刮伤涂层时, 电镀的细胞。如果细胞不附着或迁移的选择矩阵, 其他类型的涂层, 例如胶原蛋白1或纤维连接蛋白, 可以探索, 或浓度的基质蛋白, 孵化时间, 和孵化温度在涂层的板块改变。在文献中可以找到合适的细胞外基质。
斑点法有很少的局限性。这是一个二维迁移分析, 因此, 这可能被认为是一个劣势相比, 三维的分析, 然而, 更难以执行比二维化验。一个主要的问题也许是细胞增殖会影响结果, 特别是如果使用快速增殖细胞系和细胞迁移评估较长的时间段, 在这里做了4天后的菌落直径分析。同样, 在划痕的分析中, 伤口的闭合会受到细胞内细胞增殖的影响;在博伊登室的检测, 特别是如果血清或其他介质诱导细胞增生作为趋化因子, 增加细胞增殖在底部腔可以扭曲结果。单细胞跟踪可用于特别观察非增殖细胞的运动。此外, 细胞增殖可以减少的 (i) 血清饥饿, 在这里做和/或 (ii) 抑制有丝分裂的加入丝裂霉素或其他增殖抑制剂4,16。然而, 为了减少细胞增殖, 必须注意不要损害细胞, 使其无法再迁移。有趣的是, 我们发现在类似的无约束迁移分析中, 细胞增殖与边缘移位无关, 尽管它在迁移过程中涉及保持上皮片完整性4,5, 17。这就打开了一个有趣的问题: 细胞增殖是细胞表迁移的必要组成部分吗？如果完全阻断细胞的增殖, 最终将不再能够扩展和支持细胞的迁移, 那么细胞板的迁移将会对周围环境产生负面影响。此时, 单元格应减慢或停止迁移, 或者工作表应中断。反之, 由于细胞可以使床单独立迁移, 例如在上皮细胞的边缘散射, 因此, 低细胞粘连的薄片的迁移可能会受到较少的影响。因此, 细胞迁移和细胞增殖是相互依存的, 任何阻止细胞增殖的尝试都需要仔细考虑。因此, 化验分析表迁移主要是在血清饥饿, 减少, 但不取消细胞迁移。
其他迁移分析在二维表面研究板料迁移包括刮伤或创伤分析和, 最近, 无约束的迁移化验使用硅或矽氧烷室和钢板蜡纸允许电镀细胞在定义的区域和将单元格或模具1、3、4、5、6、7、16移除后, 不受约束地迁移到单元格区域。这些后检测和点分析提供了高再现性的经验和经验用户的手, 因为细胞电镀确定的形状和大小的殖民地。相反, 在刮痕的细胞单层的抓挠分析需要练习, 甚至在专家的手中, 如果使用的细胞系形成致密的单分子, 那么即使是在纸上的部分也可以被错误地撕掉。这一问题是改进时, 硅树脂室或钢板蜡纸用于电镀的细胞, 虽然我们观察到错误的伤害细胞板后去除信息处理室。斑点化验避免这个问题, 因为细胞被镀到一个殖民地, 并允许自由生长。由于同样的原因, 点分析也允许细胞迁移在不变的涂层, 这是很容易被划伤或去除的商会/模具在其他迁移化验。此外, 由于细胞不被划伤或移除室/钢板蜡纸, 细胞死亡和相关的释放细胞因子与去除室/钢板蜡纸没有影响迁移的点测定。最后, 细胞往往是在划痕化验和化验使用商会或钢板蜡纸。因此, 在清除细胞后, 通过刮掉或去除阻碍细胞迁移的物理屏障, 细胞单层就会放松, 细胞被推入细胞, 而不会主动迁移。相比之下, 在点法中, 细胞片可以在观察到迁移之前一夜之间建立, 细胞迁移而不是松弛的细胞表可以观察到。
点法, 可以很容易地执行没有任何专用设备, 借给自己的一系列后续化验, 包括细胞染色评估总菌落形态学和细胞散射8, 免疫8, 时间推移使用 ImageJ、Matlab 或其他图像分析软件13进行成像和后续数据分析。其他参数, 如相邻向量的相关性或其分离轨迹可以被评估, 以更精确地描述细胞移动的连贯性在工作表13。时间推移成像也可以结合免疫和 ImageJ 支持的细胞跟踪, 以获得进一步了解细胞迁移。例如, 在时间推移成像后, 点可以 immunostained 骨架蛋白质, 然后利用 ImageJ 来追踪细胞, 以分析这些蛋白质的表达如何影响细胞的迁移表型。另一个未来的用途是细胞的单细胞跟踪, 表达核荧光蛋白。重要的是, 点分析可以在多井板上进行, 是如此适合中-高吞吐量成像, 使它成为一个容易接近和非常实惠的工具, 以筛选药物或 shRNAs 的影响, 在床单移徙。

迁移分析被广泛用于评价肿瘤细胞的侵袭和转移潜能体外。最常见的情况是, 伤口或划痕检测是用来评估上皮细胞的迁移到一个单元清除区域1,2,3。为了进行划痕分析, 细胞被镀成单层, "划痕" 或无细胞区是用吸管尖端创建的。刮痕分析很容易建立与常用的组织培养用品, 可以进行多井板, 允许处理多个样品。然而, 当划痕时, 细胞从单层中被物理地移除, 并经常经历细胞死亡。此外, 在刮伤过程中, 细胞外基质常被破坏。同样, 使用硅刀片 (如信息处理室4) 或模具的5、6、7可以导致细胞的机械破坏和用于涂层的基质蛋白的部分去除。板.检测伤口或划痕闭合的另一个缺点是其有限的时间过程, 因为只有在抓痕闭合时才能分析细胞迁移。
在进行点检测时, 细胞被镀成一个圆形的菌落到涂层或未涂覆的平板上8。这种电镀策略的基本原理是获得具有定义边缘的细胞板, 它可以迁移或侵入周围的无细胞区, 而不会通过去除细胞或插入物而扰乱培养。斑点法的总体目标是观察细胞板的迁移, 如边缘位移或菌落直径, 以及执行时间推移成像, 以分析更高的时空分辨率的细胞移行表型。
细胞迁移可能受到各种微的影响, 如趋化因子, 细胞因子, 和生长因子, 如 EGF。egf 是通过结合其受体、egf 受体9而施加其生物效应的生长因子, 并增加肿瘤细胞的侵袭和转移行为4、9、10。在这里, 点法是用来研究 EGF 刺激细胞迁移在一个人侵入, 肺菌落形成乳腺癌细胞系 (MCF10CA1a)8,11,12。

<table style="border-collapse:collapse;width:659pt" width="878">
	<colgroup>
		<col style="width:302pt" width="402" />
		<col style="width:160pt" width="213" />
		<col style="width:131pt" width="175" />
		<col style="width:66pt" width="88" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="width:302pt;height:15.75pt" width="402">MCF10CA1a cells</td>
			<td style="width:160pt" width="213">Karmanos Research Institute</td>
			<td class="xl18" style="width:131pt" width="175">N/A</td>
			<td style="width:66pt" width="88">cells used for asay</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">mouse collagen IV</td>
			<td>BD Biosciences</td>
			<td class="xl18">354233</td>
			<td>used to coat plates</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">trypsin / EDTA</td>
			<td>Thermo Fisher</td>
			<td class="xl19">25300054</td>
			<td>used to remove cells from tissue culture plates</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">DPBS&#160;</td>
			<td>Mediatech</td>
			<td class="xl18">21-031-CV</td>
			<td>used to wash cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">DMEM / F12</td>
			<td>Thermo Fisher</td>
			<td class="xl18">11320082</td>
			<td>used as culture medium</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">horse serum</td>
			<td>Thermo Fisher</td>
			<td class="xl18">26050088</td>
			<td>used to supplement culture medium</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">12well glass bottom plate</td>
			<td>MatTek</td>
			<td class="xl18">P12G-1.5-14-F</td>
			<td>used to grow cells for imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">LPA (1-Oleoyl Lysophosphatidic Acid )</td>
			<td>Cayman</td>
			<td class="xl18">10093</td>
			<td>used to stimulate cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">EGF (epidermal growth factor, human recombinant)</td>
			<td>Peprotech</td>
			<td class="xl18">AF-100-15</td>
			<td>used to stimulate cells</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">fatty acid free BSA</td>
			<td>Sigma</td>
			<td class="xl18">A8806-1G</td>
			<td>used to make buffer for LPA and EGF</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">hematoxylin</td>
			<td>Sigma</td>
			<td class="xl18">HHS32</td>
			<td>used to stain colonies</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">eosin</td>
			<td>Electron Microscopy Sciences</td>
			<td class="xl18">26051-10</td>
			<td>used to stain colonies</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">37C, 5% CO2 incubator</td>
			<td>Sanyo</td>
			<td class="xl18">model MCO-18AIC (UV)</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Zeiss AxioObserver with incubator chamber and motorized stage</td>
			<td>Zeiss</td>
			<td class="xl18">model Axio Observer.Z1</td>
			<td>used for time lapse imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">ORCA Flash LT sCMOS camera</td>
			<td>BioVision</td>
			<td class="xl21">C11440-42U-KIT</td>
			<td>used for timelapse imaging in combination with Zeiss AxioObserver</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Metamorph</td>
			<td>BioVision</td>
			<td class="xl18">MMACQMIC</td>
			<td>software used for time lapse imaging</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">inverted microscope</td>
			<td>Olympus</td>
			<td class="xl18">model IX70</td>
			<td>used to count cells and to check colonies in tissue culture</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">plastic box</td>
			<td>Lock&#38;Lock</td>
			<td class="xl18">N/A</td>
			<td>used as humid chamber (any tight closing plastic box that fits the plates can be used)</td>
		</tr>
	</tbody>
</table>

using the dot assay to analyze migration of cell sheets

虽然复杂的有机体出现静态, 他们的组织是在连续的营业额之下。随着细胞衰老, 死亡, 并被新的细胞取代, 细胞在组织内以一种严密的方式移动。在肿瘤的发展过程中, 这种平衡受到干扰, 肿瘤细胞离开原发细胞侵入局部微环境, 前往远处的地点, 最终形成远处的转移性肿瘤。点法是一个简单的, 二维无约束迁移分析, 以评估细胞板的净移动到一个无细胞的区域, 并利用时间推移成像的细胞迁移参数。在这里, 点化验显示使用一个人侵入, 肺殖民地形成乳腺癌细胞线, MCF10CA1a, 以分析细胞的迁移反应表皮生长因子 (EGF), 这是已知的增加恶性电位的乳腺癌细胞和改变细胞的迁移表型。

这里提出的点检测 (图 1) 是利用侵入性, 肺菌落形成乳腺癌细胞 (MCF10CA1a) 作为一个模型系统进行的。点检测产生高度可重现的细胞点, 甚至在初学者的手, 使其方便, 易于执行的检测 (图 1D)。斑点分析结合 he 染色, 或时间推移成像和后续粒子图像测速 (PIV), 允许研究各种迁移参数, 包括上皮边缘的位移, 细胞速度和细胞方向性作为细胞边缘上皮片移植到无细胞区。最初, 细胞点的相衬成像显示, 侵袭性, 肺集落形成乳腺癌细胞 (MCF10CA1a) 保持间质表型的刺激后, EGF。然而, 在 EGF 刺激的细胞点 (图 2, 箭头) 中, 离开工作表的单个细胞更频繁地被观察到。MCF10CA1a 细胞对 egf 的反应在4天内对 egf 刺激的培养进行了进一步的评估。的确, EGF 刺激导致蜂群直径增加 (图 3)。这些观察表明, EGF 刺激细胞的迁移可能发生改变。
因此, 采用点法进行了 MCF10CA1a 细胞板动态迁移表型的分析。在细胞的刺激下, 片状边缘的细胞被成像9小时 (视频 1和视频 2), 然后在 Matlab 中用 PIV 进行了图像分析4,13,14。PIV 显示 EGF 增加细胞速度 (图 4A) 到0.63 µm/分钟从0.45 µm/分钟的控制单元。同时, egf 增加了细胞的方向性, 这是通过减少细胞方向性的变化 (角传播) 从0.95 在控制文化到0.79 在 EGF 被刺激的文化 (图 4B)。EGF 增加了上皮边缘的径向移位9小时从257µm 到356µm (图 4), 也观察到他染色后4天 (图 3)。因此, 使用点法结合不同的后续化验表明, EGF 改变迁移的边缘 MCF10CA1a 细胞板, 使细胞的速度和方向性增加, 从而增加了蜂群半径。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56451/56451fig1.jpg"> 图 1:成像点检测.(A, B)在玻璃底板井的中心, 细胞被镀成圆形的菌落。对于迁移分析, 图像是在两个对立的边缘以及殖民地的中心 (A) 拍摄的。(C) 在电镀过程中 (左) 的细胞点的暗场图像, 并在更高的放大倍数 (相衬) 显示的边缘的下降和边缘的细胞后, 连接到板块 (中心) 的细胞点。附加单元格是平坦的和多边形的, 而非附加的单元格是圆的 (右)。(D)点的直径是高度可再现的。在电镀后, 在细胞被刺激和使用 ImageJ 的直径确定后立即成像点。在一个实验中, 网点大小是高度可再现的, 在不同的用户之间和很少的培训, 以获得熟练的电镀细胞点 (E = 超过10年的经验, 组织培养, 我 = 少于4周的经验, 在组织 c文化)。n = 每组12点。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56451/56451fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56451/56451fig2.jpg"> 图 2:EGF 调节 MCF10CA1a 细胞的形态表型.MCF10CA1a 细胞在 DMEM/F12 补充与0.1% 马血清之前, 他们被刺激与 EGF (5 ng/毫升) 的3小时。在 EGF 刺激的菌落边缘, 单个细胞趋向于从片状 (箭头) 中迁移出来。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56451/56451fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56451/56451fig3.jpg"> 图 3: EGF 增加细胞菌落的净生长.MCF10CA1a 细胞在 DMEM/F12 培养基中以0.1% 匹马血清为 3 h, 在与 EGF (5 ng/毫升) 刺激前被补充。4天后, 细胞被乙醇固定, 用苏木精 & 红素染色。EGF-受激点在4天后有更高的直径。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56451/56451fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56451/56451fig4.jpg"> 图 4:EGF 调节 MCF10CA1a 细胞的迁移表型。MCF10CA1a 细胞在 DMEM/F12 培养基中以0.1% 匹马血清为 3 h, 在与 EGF (5 ng/毫升) 刺激前被补充。细胞被成像每3分钟9小时. 随后的粒子图像测速分析是在 Matlab13中进行的。速度值表示每个字段的平均速度。速度向量的角传播计算为: <img alt="Equation 1" src="/files/ftp_upload/56451/56451eq1.jpg">其中, 范围从 0 (高方向性) 到 (低方向性) 不等. <img alt="Equation 2" src="/files/ftp_upload/56451/56451eq2.jpg"> </img> <img alt="Equation 3" src="/files/ftp_upload/56451/56451eq3.jpg">角传播的减少被认为是方向性的增加。通过在 t = 9 h 的菌落半径上减去 0 h 的菌落半径, 计算出径向位移. EGF-MCF10CA1a 细胞点的刺激导致细胞速度增加 (A; 上部板), 角向扩散或增加方向性 (B; 中间面板), 并增加了菌落半径 (C; 底部面板)。数据代表了 n = 3 实验的平均± SEM。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56451/56451fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/56451/56451movie1.jpg"> 视频 1:刺激 MCF10CA1a 细胞的时间推移成像.每3分钟拍摄一次9小时的图片.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56451/A1-L_ctrl_Substack_(1-181-3).avi" target="_blank">请点击这里查看视频。(右键单击可下载.</a>
<img alt="Movie 2" src="/files/ftp_upload/56451/56451movie2.jpg"> 视频 2:MCF10CA1a 细胞的时间推移成像刺激 EGF (5 ng/毫升)。每3分钟拍摄一次9小时的图片.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56451/B1-L_EGF_Substack_(1-181-3).avi" target="_blank">请点击这里查看视频。(右键单击可下载.</a>

Watch this Scientific Journal Video about Using the Dot Assay to Analyze Migration of Cell Sheets at JoVE.com

Using the Dot Assay to Analyze Migration of Cell Sheets

用点法分析细胞板的迁移

细胞迁移是发展, 组织维护和修复, 和肿瘤发生的关键, 并由生长因子, 趋化因子, 和细胞因子调节。本协议描述的点法, 一个二维, 无约束的迁移分析, 以评估微提示的反应, 连接的, 有凝聚力的细胞表移表型。

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

using-the-dot-assay-to-analyze-migration-of-cell-sheets

Nanyang Technological University

Research

JoVE Journal

Cancer Research

6.8K 次观看.  National Institutes of Health. 细胞迁移是发展, 组织维护和修复, 和肿瘤发生的关键, 并由生长因子, 趋化因子, 和细胞因子调节。本协议描述的点法, 一个二维, 无约束的迁移分析, 以评估微提示的反应, 连接的, 有凝聚力的细胞表移表型。

视频: Using the Dot Assay to Analyze Migration of Cell Sheets

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

颈动脉肿瘤细胞注射，产生脑转移瘤小鼠模型

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

科研

癌症研究

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

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 ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

用球形发芽法评价 Hemangioblastoma 新生血管的体外性能的综合程序

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

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 ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...