Name: quantification of cell-substrate adhesion area and cell shape distributions in mcf7 cell monolayers
Uploaded: 2020-06-24T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers at JoVE.com

波兰国家科学中心根据第2014/14/M/NZ1/00437号赠款支持了这项工作。

1. 准备步骤
<ol><li>细胞播种，以获得汇合单层<ol>
<li>播种前，用胶原蛋白 I（或选择的其他 ECM 组件）涂覆 4 孔室滑轨的孔。对于胶原蛋白 I 涂层，请遵循https://www.sigmaaldrich.com/technical-documents/articles/biofiles/collagen-product-protocols.html：浓度为 8 μg/cm2。</li>
		<li>种子400，000细胞到一个井的4井室幻灯片。</li>
		<li>染色前培养细胞24小时（或更长，取决于实验终点），在37°C的培养箱中，5%CO2?...

<ol>
	<li>Li, D. S., Zimmermann, J., Levine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+closure+of+circular+wounds+through+coordinated+collective+motion.">Modeling closure of circular wounds through coordinated collective motion.</a> Search Results. 13 (1), 016006 (2016).</li><li>Ilina, O., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+collective+cell+migration+at+a+glance.">Mechanisms of collective cell migration at a glance.</a> Journal of Cell Science. 122, 3203-3208 (2009).</li><li>Ladoux, B., Mege, R. 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B. C., Kurniawan, N. A., Bouten, C. V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+quantitative+analysis+of+cell,+nucleus+and+focal+adhesion+morphology.">An automated quantitative analysis of cell, nucleus and focal adhesion morphology.</a> PLoS One. 13 (3), 0195201 (2018).</li><li>Fokkelman, M., 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=Cellular+adhesome+screen+identifies+critical+modulators+of+focal+adhesion+dynamics,+cellular+traction+forces+and+cell+migration+behaviour.">Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour.</a> Scientific Reports. 6, 31707 (2016).</li><li>Horzum, U., Ozdil, B., Pesen-Okvur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+quantitative+analysis+of+focal+adhesions.">Step-by-step quantitative analysis of focal adhesions.</a> MethodsX. 1, 56-59 (2014).</li><li>Kim, D. H., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+size+uniquely+predicts+cell+migration.">Focal adhesion size uniquely predicts cell migration.</a> FASEB Journal. 27 (4), 1351-1361 (2013).</li><li>Pincus, Z., Theriot, J. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Texture-based+segmentation+and+a+new+cell+shape+index+for+quantitative+analysis+of+cell+spreading+in+AFM+images.">Texture-based segmentation and a new cell shape index for quantitative analysis of cell spreading in AFM images.</a> Cytometry A. 87 (12), 1090-1100 (2015).</li><li>Tong, J., 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=Cell+micropatterning+reveals+the+modulatory+effect+of+cell+shape+on+proliferation+through+intracellular+calcium+transients.">Cell micropatterning reveals the modulatory effect of cell shape on proliferation through intracellular calcium transients.</a> Biochimica et Biophysica Acta. 1864 (12), 2389-2401 (2017).</li><li>Vartanian, K. B., Kirkpatrick, S. J., Hanson, S. R., Hinds, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+cytoskeletal+alignment+independent+of+fluid+shear+stress+on+micropatterned+surfaces.">Endothelial cell cytoskeletal alignment independent of fluid shear stress on micropatterned surfaces.</a> Biochemical and Biophysical Research Communications. 371 (4), 787-792 (2008).</li></ol>

细胞细胞和细胞基质粘附构成上皮细胞的固有属性，在组织形态生成和痛苦中起关键作用。在成人组织中，适当调节细胞层的机械特性对于维持平衡和预防肿瘤进展和转移等病理反应至关重要。焦点粘附的大小和数量取决于细胞基质粘附的强度，而细胞形状取决于收缩力，并且与细胞-细胞接触的状态有关。
在这里，我们描述了两个简单的定量分析方法，即由免疫荧光染色的?...

上皮细胞单层作为一个整体，其中细胞和细胞基板粘附以及收缩力和张力代表重要的参数,，其适当的平衡有助于单元1，2，3,2的整体完整性。因此，评估这些参数是建立细胞层的当前状态的一种方式。
此处描述的两种方法表示对粘附的上皮细胞（本例中为 MCF7 乳腺癌细胞系）的汇合单层进行二维分析。使用 Z 轴上不同区域的共和图像（单个 Z 切片）执行分析;基座附近的基底区域，用于焦粘附 （FA） 测量，用于细胞形状测量的平面区域。提出的方法相对简单，需要标准的实验室技术和开源软件。共体显微镜足以满足此协议，因此无需采用更专业的 TIRF（总内部反射荧光）显微镜即可执行。因此，该议定书可以在相对标准的实验室环境中实施。虽然方法的准确性有限，但它们可以区分焦粘附和细胞形状的基本差异。
此处描述的两种方法都包括使用 ImageJ 执行的标准实验程序，如细胞培养、免疫污染、共体成像和图像分析。但是，可以使用任何具有适当功能的图像处理软件。提出的方法可以跟踪和比较药理治疗或最小的基因改造带来的变化。由于这些方法的精度有限，不建议获取确定值。包括两个自动宏，以方便测量许多图像。

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A32740</td>
			<td>goat anti-rabbit, 1:500</td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>BioShop</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from calf skin</td>
			<td>Sigma</td>
			<td>C9791-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1:10000 (stock 1 mg/mL in H2O), nucleic acid staining</td>
		</tr>
		<tr>
			<td>DMEM + GlutaMAX, 1 g/L D-Glucose, Pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10270-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Junction plakoglobin</td>
			<td>Cell Signaling</td>
			<td>2309S</td>
			<td>rabbit, 1:400</td>
		</tr>
		<tr>
			<td>Laminar-flow cabinet class 2</td>
			<td>Alpina</td>
			<td></td>
			<td>standard equipment</td>
		</tr>
		<tr>
			<td>MCF7-basedHAX1KD cell line</td>
			<td>Cell line established in the National Institute of Oncology, Warsaw, described in Balcerak et al., 2019</td>
			<td></td>
			<td>MCF7 cell line withHAX1knockdown</td>
		</tr>
		<tr>
			<td>MCF7 cell line (CONTROL)</td>
			<td>ATCC</td>
			<td>ATCC HTB-22</td>
			<td>epithelial, adherent breast cancer cell line</td>
		</tr>
		<tr>
			<td>Olympus CK2 light microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paxillin</td>
			<td>Abcam</td>
			<td>ab32084</td>
			<td>rabbit, 1:250, Y113</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-TRITC conjugate</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>1:400 (stock 5 mg/mL in DMSO), actin labeling</td>
		</tr>
		<tr>
			<td>PTX</td>
			<td>Sigma</td>
			<td>T7402-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TBST &#8211; NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>TBST &#8211; Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM800 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of cell-substrate adhesion area and cell shape distributions in mcf7 cell monolayers

此处介绍的方法从多个适当染色的共聚焦图像中量化了汇合粘附细胞单层之间的一些参数：粘附到基底，作为焦粘附数和大小的函数，以及以细胞形状索引和其他形状描述符为特征的细胞形状。焦粘附物通过paxillin染色和细胞边界以结球蛋白和行动素为标志。细胞培养和染色的方法是标准的;图像代表单个焦点平面;使用公开提供的图像处理软件进行图像分析。提出的协议用于量化焦点粘附的数量和大小以及单层中细胞形状分布的差异，但它们可以重新用于量化任何其他可染色的不同细胞结构（例如线粒体或核）的大小和形状。评估这些参数对于粘附细胞层中动态力的表征非常重要，包括影响细胞形状的细胞粘附和运动素收缩性。

聚焦粘附分析 HAX1基因的敲击先前被证明会影响焦点粘附6。细胞在胶原蛋白 I 涂层表面培养 48 小时。MCF7 控制细胞和 MCF7 细胞的图像与HAX1击倒（HAX1 KD） 从三个独立的实验染色的焦点粘附蛋白 paxillin 获得使用共聚焦显微镜 （图像从单焦平面/Z 切片从基础区域）.使用所述协议对来自每个细胞系中的大约 2，000-2，500 个细胞的 FA 进行量化。最小?...

Watch this Scientific Journal Video about Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers at JoVE.com

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

本文描述了1）焦点粘附的大小和数量，2）细胞形状指数及其分布从MCF7细胞的汇合单层共聚焦图像。

MCF7细胞单层中细胞基质粘附面积和细胞形状分布的定量

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

该协议允许从二维图像中量化细胞内结构的面积、周长和形状。这种技术是快速和容易的，只需要标准的实验室设置和共和显微镜，并使用开源软件。演示这个程序的将是安娜·巴尔塞拉克，一个博士学生从我的实验室。首先，在四井胶原蛋白一个涂层室幻灯片中，每井在 0.5 毫升培养培养基中播种四倍至第五细胞，在 37 摄氏度和 5 度二氧化碳培养箱中进行 24 小时培养。第二天早上，使用光学倒置显微镜验证是否存在至少90%的汇合单层，并使用共和显微镜获得单片Z切片图像。对于焦距粘附分析，请在 ImageJ 中打开图像并选择分析、设置比例、移除比例和全局以像素为单位设置图像比例。若要在测量选项中包括文件名和感兴趣区域的区域，请单击分析和设置测量值，并检查区域并显示标签选项。要减去背景，请选择过程并减去背景。将滚动球半径设置为 50 像素，并检查滑动抛物线。要确定最小感兴趣区域的区域，请勾勒出最小的单个焦点粘附，然后单击分析和测量以测量该区域。当至少选择并测量了 20 个感兴趣区域时，计算并保存获得的结果的均值。将上边界设置为典型单元格区域的 25%。若要将图像授予二进制图像，请单击图像、调整和阈值，并检查默认、黑白和深色背景。要测量感兴趣区域的数量和面积，请选择分析粒子并检查像素单位、显示结果、明确结果和汇总。将切片、计数和总面积平均大小数据从摘要窗口传输到所选的数据管理程序。对于焦粘附定量，打开焦粘附 ImageJ 宏，进入最小感兴趣区域的区域作为最小感兴趣区域参数的区域。将阈值类型值设置为手动或自动，并保存更改。然后从 ImageJ 调用宏并选择要处理的图像。对于手动单元格形状分析，请在适当的图像处理软件程序中打开图像，然后选择要测量的参数。使用徒手选择工具手动划定由所选的结蛋白标记的细胞边框。将自动计算每个单元格的选定参数。概述所有单元格后，选择编辑、选择并添加到管理器。只应选择具有不间断边框的完整完全可见的单元格。要进行测量，请标记兴趣管理器区域左侧框中显示的所有数字，然后单击"度量"。结果将显示在结果框中，并可以导入到首选电子表格。自动细胞分析有助于定量大量细胞。对于每个新的单元格类型，首先运行宏以建立参数。宏完成后，选择设置单元格大小边界。单击最小和最大单元格的标签，然后单击度量值。设置最小单元格和最大单元格变量的值并保存更改。关闭所有宏窗口，然后选择要以灰度处理的图像。然后再次运行宏。宏将提供一个结果表，其中包括单元格形状索引、纵横比和兴趣选择区域列表数据。在这里，可以观察到与 ImageJ 一起计数的焦粘附的代表性图像，包括最终编号的轮廓和焦粘附轮廓与原始图像的叠加，用于控制和击倒细胞系。如本分析所示，与控制细胞系细胞相比，击倒细胞表明每个细胞的粘附数以及较大的粘附量。在这些图像中，显示了未经治疗和化疗的细胞单层代表性区域。轮廓细胞的特征是按照其细胞形状指数值，例如，在此分析中，与未经治疗的对控相比，显示最后一个 bin 的增加和药物治疗细胞的主要峰值的扁平化。然后可以为每个单元培养生成频率分布和累积分布。所演示的单层灰度成像允许对来自12个视场的512个细胞进行分析和定量，揭示细胞形状指数的相对频率分布。对于此协议来说，使用尽可能高质量的图像非常重要。适当的细胞播种、染色和成像应确保图像具有足够的实验质量。

Research

JoVE Journal

Biology

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

In situ Quantification of Pancreatic Beta-cell Mass in Mice

In situ Quantification of Pancreatic Beta-cell Mass in Mice

在小鼠胰腺β-细胞团的原位定量

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

分析细胞表面粘附重塑回应机械张力使用磁珠

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Automating Aggregate Quantification in Caenorhabditis elegans

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...