Name: examining the dynamics of cellular adhesion and spreading of epithelial cells on fibronectin during oxidative stress
Uploaded: 2019-10-13T12:30:00
Description: Watch this Scientific Journal Video about Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress at JoVE.com

作者感谢斯科特·赫顿博士和梅根·布莱克利奇博士对手稿的批判性评论。这项工作由高点大学的研究和赞助项目（MCS）和北卡罗来纳州立大学生物技术项目资助。

1. 准备工作
注： 下面描述的协议已针对 REF52 细胞和 ATM+/*或 ATM-/-人成纤维细胞的使用进行了优化。其他单元格类型可能需要进一步优化，如下面的说明和故障排除部分所述。
<ol><li>为REF52细胞制作500 mL的完整细胞培养基。在含有Dulbeco的改性鹰培养基（DMEM）的500mL中，可添加10%FBS、2 mM L-谷氨酰胺和100单位/mL青霉素-链霉素。</li>
	<li>在12mL无菌1x磷酸盐缓冲盐水...

<ol>
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T., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoA+inactivation+by+p190RhoGAP+regulates+cell+spreading+and+migration+by+promoting+membrane+protrusion+and+polarity.">RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity.</a> Molecular Biology of the Cell. 12 (9), 2711-2720 (2001).</li><li>Chandra, S., Kalaivani, R., Kumar, M., Srinivasan, N., Sarkar, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+recruits+cellular+villin+to+remodel+actin+cytoskeleton+during+fusion+with+hepatocytes.">Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes.</a> Molecular Biology of the Cell. 28 (26), 3801-3814 (2017).</li><li>Fitzpatrick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Measuring Cell Fluorescence Using ImageJ. , (2014).</li><li>Berginski, M. E., Vitriol, E. A., Hahn, K. M., Gomez, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+quantification+of+focal+adhesion+spatiotemporal+dynamics+in+living+cells.">High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells.</a> PLoS One. 6 (7), e22025 (2011).</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>Elosegui-Artola, A., 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=Image+analysis+for+the+quantitative+comparison+of+stress+fibers+and+focal+adhesions.">Image analysis for the quantitative comparison of stress fibers and focal adhesions.</a> PLoS One. 9 (9), e107393 (2014).</li><li>Meller, J., Vidali, L., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+RhoG+is+dispensable+for+integrin-mediated+cell+spreading+but+contributes+to+Rac-independent+migration.">Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration.</a> Journal of Cell Science. 121 (Pt 12), 1981-1989 (2008).</li><li>Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+Staining.">Immunofluorescence Staining.</a> Current Protocols in Cell Biology. 69 (43), 1-7 (2015).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunocytochemistry:+an+update.">Controls for immunocytochemistry: an update.</a> Journal of Histochemistry and Cytochemistry. 59 (1), 6-12 (2011).</li><li>Grashoff, 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=Measuring+mechanical+tension+across+vinculin+reveals+regulation+of+focal+adhesion+dynamics.">Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.</a> Nature. 466 (7303), 263-266 (2010).</li><li>Kumar, A., 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=Correction:+Talin+tension+sensor+reveals+novel+features+of+focal+adhesion+force+transmission+and+mechanosensitivity.">Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.</a> Journal of Cell Biology. 214 (2), 231 (2016).</li><li>Kumar, A., 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=Talin+tension+sensor+reveals+novel+features+of+focal+adhesion+force+transmission+and+mechanosensitivity.">Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.</a> Journal of Cell Biology. 213 (3), 371-383 (2016).</li><li>Friedrichs, J., Helenius, J., Muller, D. 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此处描述的协议是一种通用且经济的方法，可快速筛选多个与锚固相关的细胞类型，以便在细胞扩散期间进行动态细胞骨架重塑。特别是，当细胞粘附在FN上时，这种方法定量地检查氧化应激期间的应力纤维和焦点粘附形成（图1A）。此外，这些细胞表型可能建议一个监管作用的Rho家族的小GTPases的成员，因为他们有记录的作用，在细胞附件和传播15，16，22。</sup...

细胞基质粘附（即焦点粘附）是大而动态的多分子蛋白复合物，可调节细胞粘附和扩散。这些过程对于组织发育、维护和生理功能至关重要。焦点粘附由膜结合受体（如整数）以及将细胞骨骼功能素与细胞外基质（ECM）1联系起来的脚手架蛋白组成。这些复合物能够通过激活各种信号转导途径来响应细胞外环境中的物理化学线索。因此，焦点粘附作为信号中心，将细胞外机械线索传播到许多细胞过程，包括定向迁移、细胞周期调节、分化和存活1、2。一组调节焦点粘附并与之相互作用的信号分子包括小GTPases的Rho家族的成员。Rho GTPass是关键的蛋白质，通过它们特定的时空活化3调节细胞迁移和粘附动力学。毫不奇怪，Rho蛋白功能调节障碍与一些人类病理有关，如转移、血管生成等。特别令人感兴趣的是，细胞氧化还原状态在细胞迁移和粘附的调制中起着主要作用。氧化还原平衡的改变，如活性氧物种（ROS）的增加，已被证明可以调节许多细胞类型和人类疾病中的Rho蛋白活性和附着力4、5、6 ，7，8.例如，患有神经紊乱（A-T），这是由DNA损伤修复丝氨酸/三甲氨酸激酶A-T突变（ATM）的突变引起的，转移性癌症的风险增加9， 10.这些患者和细胞系的ATM激酶活性丧失，无论是通过基因突变还是化学抑制，导致由于磷酸苯甲酸酯通路7、11的功能障碍而产生高水平的氧化应激。 12.此外，实验室最近的研究通过改变细胞骨骼动力学（即粘附和扩散）在A-T中对ROS的病理生理学作用，直接导致在体外激活Rho家族GTPases。最终，由Rho家族活化引起的细胞骨骼动力学的这些改变可能导致A-T患者5、13中注意到的转移性癌症风险增加。因此，了解氧化应激期间细胞-基质相互作用之间的相互作用，可以深入了解粘附和扩散的调节。这些研究还可以为进一步调查 Rho 家族 GTPass 在这些信号过程中可能扮演的角色设置舞台。
本文所述是研究ATM激酶活性抑制引起的氧化应激期间粘附组装和扩散的早期细胞动力学的一种方案。此测定基于锚固依赖细胞粘附于 ECM 蛋白纤维蛋白 （FN） 的良好特征机制。当悬浮细胞被镀在FN上时，几个Rho GTPass协调对细胞骨骼重塑的控制3，14。当细胞从外观的圆形和圆形向扁平和膨胀移动时，形态变化被观察。伴随这些观察的是与 ECM 一起开发许多矩阵粘附。这些变化归因于当细胞粘附并扩散15、16时，在1小时内用Rac1对RhoA进行双相激活。
已采用各种方法检查粘附形态和动力学以及细胞扩散。然而，这些方法依赖于复杂的长期实时成像全内反射荧光（TIRF）或共聚焦显微镜系统。因此，用户必须有权访问专用设备和软件。此外，这些生物成像系统所需的设置时间使得捕获早期粘附事件具有挑战性，尤其是在同时测试多种抑制剂或治疗条件时。
本文详细的方法提供了一种简单、经济、定量的方法来评估控制粘附组件和体外扩散的参数。该协议使用常用的实验室设备执行，如荧光显微镜和CCD相机。这种测定涉及在ATM激酶活性化学抑制引起一段时间的氧化应激后，将锚固依赖细胞应用于FN涂层表面，这在之前已经证明了这一点。电镀后，允许电池在指定的时间长度上附加和粘附。未附着的细胞被冲走，而附着的细胞被固定，并标有荧光基抗体的附着力（例如，帕西林）和扩散（例如，F-actin）2，5 。然后，这些蛋白质使用荧光显微镜进行可视化和记录。随后的数据分析使用免费提供的斐济软件进行。此外，该方法可以适应，以检查粘附动力学在广泛的条件下，包括不同的ECM蛋白，处理各种氧化剂/细胞培养条件或各种锚固依赖细胞系，以解决广泛的范围生物问题。

<table>
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300-054</td>
			<td>cell dissociation</td>
		</tr>
		<tr>
			<td>10 cm2 dishes</td>
			<td>Cell Treat</td>
			<td>229620</td>
			<td>sterile, tissue culture treated</td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>1X Phosphate Buffered Saline</td>
			<td>Corning Cellgro</td>
			<td>21-031-CV</td>
			<td>PBS, sterile, free of Mg2+ and Ca2+</td>
		</tr>
		<tr>
			<td>24-well cell culture treated plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-740</td>
			<td>sterile, tissue culture treated</td>
		</tr>
		<tr>
			<td>4&#176;C refrigerator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG anti-paxillin primary antibody (clone 165)</td>
			<td>BD Transduction Laboratories</td>
			<td>610620</td>
			<td>marker of focal adhesions</td>
		</tr>
		<tr>
			<td>Aspirator</td>
			<td>Argos</td>
			<td>EV310</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet</td>
			<td>Nuair</td>
			<td>NU-477-400</td>
			<td>Class II, Type A, series 5</td>
		</tr>
		<tr>
			<td>Delipidated Bovine Serum Albumin (Fatty Acid Free) Powder</td>
			<td>Fisher Scientific</td>
			<td>BP9704-100</td>
			<td>dlBSA</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td>organic solvent to dissolve Ku55933</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Media, High Glucose</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>REF52 base cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>16000044</td>
			<td>certified, cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td>National Institutes of Health</td>
			<td>http://fiji.sc/</td>
			<td>image analysis program</td>
		</tr>
		<tr>
			<td>Filter syringe</td>
			<td>Fisher Scientific</td>
			<td>6900-2502</td>
			<td>0.2 &#181;M, sterile</td>
		</tr>
		<tr>
			<td>Glass coverslips (12-Cir-1.5)</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td>autoclave in foil to sterilize</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG secondary antibody Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>fluorescent secondary antibody, light sensitive</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Gibco by Life Technologies</td>
			<td>16210-064</td>
			<td>component of blocking solution for immunofluorescence</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>22-600-107</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin</td>
			<td>Gibco by Life Technologies</td>
			<td>33016-015</td>
			<td>FN</td>
		</tr>
		<tr>
			<td>IX73 Fluorescence Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>microscope to visualize fluorescence, cell morphology, counting and dissociation</td>
		</tr>
		<tr>
			<td>Ku55933</td>
			<td>Sigma-Aldrich</td>
			<td>SML1109-25MG</td>
			<td>ATM kinase inhibitor, inducer of reactive oxygen species</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Fisher Scientific</td>
			<td>25-030-081</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Monochrome CMOS 16 bit camera</td>
			<td>Optimos</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148-500G</td>
			<td>PFA, fixative for immunofluorescence</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td>P/S, antibiotic solution for culture medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 phalloidin (F-actin probe)</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>marker of F-actin, light sensitive</td>
		</tr>
		<tr>
			<td>ProLong Gold Anti-fade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36941</td>
			<td>cover slip mounting media including nuclear dye DAPI, light sensitive</td>
		</tr>
		<tr>
			<td>REF52 cells</td>
			<td></td>
			<td></td>
			<td>Graham, D.M. et. al. Journal of Cell Biology 2018</td>
		</tr>
		<tr>
			<td>Stir plate with heat control</td>
			<td>Corning Incorporated</td>
			<td>PC-420D</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Biosciences</td>
			<td>309653</td>
			<td>60 mL syringe</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Nuair</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>detergent used to permeabilize cell membranes</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Fisher Scientific</td>
			<td>15-250-061</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution (1x)</td>
			<td>Gibco by Life Technologies</td>
			<td>R-002-100</td>
			<td>TNS, neutralizes trypsin instead of fetal bovine serum</td>
		</tr>
		<tr>
			<td>tube rotator</td>
			<td>Fisher Scientific</td>
			<td>11-676-341</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>Fisher Scientific</td>
			<td>FSGPD02</td>
			<td></td>
		</tr>
	</tbody>
</table>

examining the dynamics of cellular adhesion and spreading of epithelial cells on fibronectin during oxidative stress

细胞粘附和扩散到细胞外基质（ECM）是有机体发育和成人组织平衡过程中必不可少的细胞过程。有趣的是，氧化应激可以改变这些过程，从而促进转移性癌症等疾病的病理生理学。因此，了解在氧化还原状态扰动期间细胞如何附着和在ECM上传播的机制，可以深入了解正常和疾病状态。下面描述是一个循序式方案，它利用基于免疫荧光的测定来具体量化细胞粘附和在体外纤维素（FN）上传播不朽的成纤维细胞。简单地说，锚固依赖细胞被悬浮，并暴露于ATM激酶抑制剂Ku55933诱导氧化应激。然后，在 FN 涂层表面上镀层，并允许在预定的时间段内附着。仍然附着的细胞是固定的，并标有荧光基抗体标记的附着力（例如，白辛）和扩散（例如，F-actin）。数据采集和分析使用常用的实验室设备进行，包括荧光显微镜和免费提供的斐济软件。此过程用途广泛，可针对各种细胞系、ECM 蛋白或抑制剂进行修改，以检查广泛的生物学问题。

实验设置的一般架构
图1显示了从REF52细胞血清饥饿开始，以获得荧光图像的计算分析结束的细胞粘附和扩散协议的一般架构。时间线中说明了协议中的关键步骤。值得注意的是，协议的第 2 步描述了 FN 涂层盖玻片的制备，该表应与步骤 3 同时进行：血清饥饿的 REF52 细胞在将其置于悬浮状态之前（图 1A）。在荧光显微镜样品固定之?...

Watch this Scientific Journal Video about Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress at JoVE.com

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

该方法可用于量化细胞粘附的早期动力学，并将锚固依赖细胞扩散到纤维质。此外，此测定可用于研究改变的氧化还原平衡对细胞扩散和/或细胞粘附相关细胞内信号通路的影响。

在氧化应激期间细胞粘附和纤维蛋白扩散动力学

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

这种方法将使研究人员能够量化细胞粘附的早期动力学，并在氧化应激期间将锚状依赖细胞扩散到细胞外基质蛋白纤维素上。该技术用途广泛，可适应于在多种条件下检查细胞骨骼动力学。此外，数据采集和分析使用通用实验室设备和免费软件进行。了解细胞在改性氧化还原状态期间如何连接和扩散 ECM 的机制，可以提供有关正常和疾病状态（如转移性癌症）的宝贵见解。此方法可用于检查不同细胞培养条件下的细胞扩散和粘附、锚系依赖细胞系和/氧化剂，以回答广泛的生物学问题。执行此技术需要大量试剂。因此，所有解决方案在开始前必须提前提出。在经过 BSL-II 认证的层流罩中，设置了组织培养认证的 24 井板。将一个玻璃盖玻片放入每一个井中。根据文本协议的图 1B 标记板。接下来，移液器500微升的纤维素溶液进入每一个井。在每个盖玻片上移液几次，以确保均匀的涂层和完全的淹没。用盖子盖住盘子。在37摄氏度下用5%的二氧化碳孵育一小时。然后从培养箱中取出板，从井中吸出纤维素溶液。每次洗涤使用 500 微升 1X PBS 洗井三次。吸走 1X PBS 的最终洗涤，并在 37 摄氏度下用 5% 的 0.5% 脱脂 BSA 溶液用 500 微升的 500 微升堵塞油井，至少 15 分钟使用 5% 的二氧化碳。首先，在37摄氏度的水浴中预热所需的溶液。在经过 BSL-II 认证的层流罩中，从 10 平方厘米培养皿中的 REF52 细胞汇合单层开始，用 6 毫升热 1X PBS 洗涤细胞两次。血清在六毫升的温暖脱脂 BSA 溶液中使细胞挨饿至少一小时。接下来，用6毫升加热的1XPBS清洗细胞。吸气PBS，并添加1.5毫升热0.5%的三辛-EDTA溶液。用5%的二氧化碳在37摄氏度下孵育细胞约两分钟。在光学显微镜下观察细胞，确保分离完成。如果细胞在敲击工作台顶部的板后仍粘附，请将它们返回到培养箱中再多两分钟。移液 1.5 毫升热尝试素中和溶液 TNS， 到菜停止尝试性， 并收集分离细胞.多次在板底上上下移液，以去除所有剩余的粘附细胞。如果细胞出现团状，则通过轻轻在盘子背面上下移液，进一步三角化细胞悬浮液。接下来，在自动单元格计数器中使用 trypan 蓝色排除数单元格计数。去除适当数量的细胞，在15毫升锥形管中，在脱脂BSA中，细胞密度在10，000至30，000个细胞之间，创建细胞悬浮液。在桌面离心机中使用固定角转子，以 300 次 g 离心电池，5 分钟。吸上清液，并在7毫升热脱脂BSA溶液中重新悬浮细胞颗粒。然后均匀地将电池悬架分成两个 15 毫升锥形管，一个用于车辆单独控制，另一个用于 Ku55933。使用管旋转器，在37摄氏度的细胞培养箱中旋转管子90至120分钟。电镀前30分钟，将Ku55933和DMSO添加到每个管中，最终浓度为10微摩尔。将电池悬架返回旋转器剩余时间。在电镀细胞之前，立即从培养箱中取出板，并吸进脱脂的 BSA 溶液。在此之后，从每个治疗组取出500微升的细胞悬浮液，并将每个微升添加到24井板中一个纤维素涂层盖滑。在以前的条件下继续孵育板和电池悬浮液。然后让单元格在所需时间长度内粘附在盖玻片上。在所需的粘附时间经过后，从板中的每个盖玻片中吸出细胞溶液。通过移液下井两侧，轻轻将 500 微升 3.7% 的半成醛溶液喷涂到每个盖玻片上，等待 10 到 15 分钟。接下来，去除甲醛溶液，每次洗涤使用 500 微升 PBS 用 1X PBS 清洗每个盖玻片两次。用 500 微升 0.2%Triton X-100 和 1X PBS 在室温下吸气 PBS 和渗透细胞 10 至 15 分钟。然后用 1X PBS 洗涤每个盖玻片三次，每次洗涤使用 500 微升 PBS。用500微升的免疫荧光阻断缓冲液阻断缓冲液阻断每个盖玻片上的细胞，30至60分钟。以1比250的比例稀释阻断缓冲液中的原发性抗多西林抗体。混合好，并添加200微升的抗体溶液到每个盖玻片。在室温下孵育至少一小时。在此之后，吸抗体溶液，用500微升1X PBS清洗每个盖玻片10分钟。保护样品从这一点向前光。稀释与红色荧光Alexa 594染料和山羊抗小鼠488荧光二次抗体结合的法罗酮F-actin探针， 在同一阻尼缓冲液溶液中。混合好，将抗体溶液的200微升添加到每个盖玻片中30分钟。吸抗体溶液，用500微升1X PBS清洗每个盖玻片10分钟。吸气 PBS 并用 500 微升去压水冲洗盖滑。然后使用包含 DAPI 的防褪色安装介质将盖滑到显微镜幻灯片上。使用抗乳素抗体固定和染色后，获得REF52细胞的荧光8位灰度图像。在完成所有图像处理步骤后，各个焦点粘附应突出、聚焦，并且易于区分。反白西林和法洛丁F-actin探针染色细胞的灰度荧光图像在镀上纤维素后被拍摄。突出的焦粘附和应力纤维在REF52细胞中应容易可见，因为允许在纤维素上粘附20至30分钟。细胞膜前缘的F-actin富集褶皱用箭头表示。分析了粉状素F-actin探针和抗白西林染色的类似荧光图像，以寻找应激纤维在细胞扩散中的百分比。值得注意的是，氧化剂治疗导致压力纤维形成显著增加，在所有粘附时间点检查，和细胞扩散后15分钟的细胞粘附到裂开素减少。以较高的细胞密度电镀会导致细胞拥挤，从而禁止细胞因过度协调而完全扩散。请注意，单元格边缘与相邻单元格无法区分。因此，单个细胞的定量被排除，并且无法准确确定周长的传播。小鼠胚胎成纤维细胞的单独细胞系被悬浮，然后镀在纤维素上30分钟。然后，细胞被固定并染色为抗白西林抗体。请注意焦点外单元格。此外，抗多西林抗体与细胞碎片的交叉反应将改变定量图像分析期间的阈值，不应包含在分析中。在图像处理过程中，使用斐济的查找工具检查图像直方图，同时调整亮度/对比度，以避免与信号饱和相关的陷阱。粘附和扩散的变化可能会影响细胞迁移。跟踪单细胞迁移、伤口愈合或化学入侵分析的方法可以确定迁移中是否发生改变。Rho 系列 GTPases 通过特定的空间时间激活来调节细胞粘附动力学。在氧化应激期间粘附和扩散的表型变化可能暗示了这些蛋白质的下游调节作用。这种方法需要使用BSL-II细胞系和危险化学试剂。研究人员需要由其机构的环境健康和安全办公室确定的化学和生物安全培训。

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

生物素化细胞表面蛋白的纯化<emRigicephalus microplus</em&gt;上皮细胞

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Evaluation of the Impact of Protein Aggregation on Cellular Oxidative Stress in Yeast

蛋白质聚集对酵母细胞氧化应激的影响评价

Protein misfolding and aggregation into amyloid conformations have been related to the onset and progression of several neurodegenerative diseases. ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

用石英谐振器研究磷脂结合蛋白附件 a2 与固体支持的脂质层结合动力学的耗散微重法

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

使用莱迪思光片显微镜可视化表面 T-Cell 受体动力学四维

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...