Name: protrusion force microscopy: a method to quantify forces developed by cell protrusions
Uploaded: 2018-06-16T14:00:00
Description: Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

作者感谢安娜 Labernadie, 纪尧姆 Charrière 和帕特里克 Delobelle 为这项工作的最初贡献, 并马修桑切斯和弗朗索瓦斯·汉普森 Viala 为他们的帮助, 视频拍摄和编辑。这项工作得到了研究所 (ANR14-CE11-0020-02)、la 基金会研究所 Médicale (FRM DEQ2016 0334894)、INSERM 计划癌症、图卢兹癌症和人类前沿科学计划 (RGP0035/2016) 的支持。

1. Formvar 涂层网格的制备
<ol><li>清洁电子显微镜网格与纯丙酮和干燥他们在滤纸上。然后用纯乙醇清洁显微镜滑梯, 用透镜纸擦拭, 用鼓风机清除灰尘。</li>
	<li>将乙醇清洗过的玻璃垂直放在薄膜浇铸装置的漏斗上, 其中含有下部 Formvar 的溶液。覆盖漏斗顶部。</li>
	<li>将100毫升的 Formvar 溶液 (二氯化乙烯中的 0.5%) 与雾化灯泡泵浦, 直到水平达到滑动的两个以上。</li>
	<li>在解决方案中保留幻灯片1分...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Elasticity+Directs+Stem+Cell+Lineage+Specification.">Matrix Elasticity Directs Stem Cell Lineage Specification.</a> Cell. 126, 677-689 (2006).</li><li>Gilbert, P. M., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Adaptation+to+Biomechanical+Stress+across+Length+Scales+in+Tissue+Homeostasis+and+Disease.">Cellular Adaptation to Biomechanical Stress across Length Scales in Tissue Homeostasis and Disease.</a> Seminars in Cell &amp; Developmental Biology. 67, 141-152 (2017).</li><li>Vining, K. H., Mooney, D. 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Force Generation and Mechanosensing Activity of Human Macrophage Podosomes.</a> Nature Communications. 5, 5343 (2014).</li><li>Proag, 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=Working+Together:+Spatial+Synchrony+in+the+Force+and+Actin+Dynamics+of+Podosome+First+Neighbors.">Working Together: Spatial Synchrony in the Force and Actin Dynamics of Podosome First Neighbors.</a> ACS Nano. 9, 3800-3813 (2015).</li><li>Proag, A., Bouissou, A., Vieu, C., Maridonneau-Parini, I., Poincloux, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Force+and+Spatial+Dynamics+of+Macrophage+Podosomes+by+Multi-Particle+Tracking.">Evaluation of the Force and Spatial Dynamics of Macrophage Podosomes by Multi-Particle Tracking.</a> Methods. 94, 75-84 (2016).</li><li>Cougoule, C., et al. <a 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V., 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=Transcellular+Diapedesis+Is+Initiated+by+Invasive+Podosomes.">Transcellular Diapedesis Is Initiated by Invasive Podosomes.</a> Immunity. 26, 784-797 (2007).</li><li>Sage, P. T., 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=Antigen+Recognition+Is+Facilitated+by+Invadosome-Like+Protrusions+Formed+by+Memory/Effector+T+Cells.">Antigen Recognition Is Facilitated by Invadosome-Like Protrusions Formed by Memory/Effector T Cells.</a> Journal of Immunology. 188, 3686-3699 (2012).</li><li>Sens, K. L., 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=An+Invasive+Podosome-Like+Structure+Promotes+Fusion+Pore+Formation+During+Myoblast+Fusion.">An Invasive Podosome-Like Structure Promotes Fusion Pore Formation During Myoblast Fusion.</a> Journal of Cell Biology. 191, 1013-1027 (2010).</li><li>Takito, 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=The+Transient+Appearance+of+Zipper-Like+Actin+Superstructures+During+the+Fusion+of+Osteoclasts.">The Transient Appearance of Zipper-Like Actin Superstructures During the Fusion of Osteoclasts.</a> Journal of Cell Science. 125, 662-672 (2012).</li><li>Shilagardi, K., 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=Actin-Propelled+Invasive+Membrane+Protrusions+Promote+Fusogenic+Protein+Engagement+During+Cell-Cell+Fusion.">Actin-Propelled Invasive Membrane Protrusions Promote Fusogenic Protein Engagement During Cell-Cell Fusion.</a> Science. 340, 359-363 (2013).</li><li>Freeman, S. 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材料性能
可变形膜材料的选择, 在我们的案例中 Formvar, 需要满足一些要求。该材料必须是透明的可见光和展示有限的自动荧光, 以允许观察在明亮的领域和荧光显微镜。薄膜的粗糙度必须低于10纳米, 以避免任何地形对细胞黏附的影响, 并允许通过 AFM 成像清晰地观察细胞诱发的突起。最后, 根据杨氏模量和材料厚度的不同, 膜的刚度需要足够高, 以诱导 podosomes 的形...

动物细胞与基质和构成其环境的其他细胞在物理上相互作用1。这是需要他们迁移, 内化机构, 获取外部信息, 或区分。在这种过程中, 细胞必须产生机械力, 正如近年来许多研究表明的那样, 细胞产生力量和探测其环境的能力会影响其生物学行为, 例如扩散或分化2,3。反过来, 细胞力的测量是研究力量生成规律的主要辅助因素, 并了解它在细胞行为和组织命运4,5中的意义。
最近几年目睹了许多技术的发展, 以测量细胞在其环境中所能发挥的力量6。大多数这些都有助于揭示细胞在移动探针或变形基板上施加的牵引力。然而, 在细胞外环境中所涉及的机械力缺乏测量技术, 至今尚无良好的特征。
为了克服这一限制, 我们提出了一种测量施加正交到基体上的力的方法。它包括在一个薄的弹性薄板上的电镀活细胞, 可以在正交方向上变形, 使其能够测量基底变形的细胞和推断所涉及的力量。用原子力显微镜测量基片的形貌, 用纳米尺度的分辨率测定, 变形力的评估依赖于突出细胞结构的几何知识7,8,9。
在这里, 我们描述的设置和它的应用, 以测量 podosomes 产生的力量, 突出黏附结构形成的巨噬细胞为他们的间充质迁移在三维环境10,11, 12,13,14,15,16,17。我们相信, 这一技术将促进理解的力量产生和它的调节, 在许多细胞过程涉及突出。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">200 mesh nickel grids</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">G200-Ni</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Filter paper</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">1001-055</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Microscope slides</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">10235612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White stickers 26 x 70 mm</td>
			<td style="width:71px;">Avery</td>
			<td style="width:119px;">DP033-100</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Film casting device with valve in its outlet</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71305-01</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Razorblades</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">72000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">1.08543.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetone</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">20066.321</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Formvar 0.5% solution in ethylene dichloride</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">12 mm coverslips</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">631-0666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Axiovert 200</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Atomic Force Microscope</td>
			<td style="width:71px;">JPK Instruments</td>
			<td style="width:119px;">NanoWizard III</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">Temperature-controlled sample holder&#160;</td>
			<td style="width:71px;">JPK Instruments</td>
			<td style="width:119px;">BioCell</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td>
			<td style="width:71px;">Veeco Instruments</td>
			<td style="width:119px;">MLCT-AUHW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double-sided adhesive tape</td>
			<td style="width:71px;">APLI AGIPA</td>
			<td style="width:119px;">118100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31870-025</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">FCS</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7524</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">HEPES&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom Petri dishes</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

在许多生物学环境中, 动物细胞需要通过培养机械力来与环境相互作用。其中, 牵引力具有良好的特征, 但缺乏技术允许测量正交细胞所施加的凸出力。我们设计了一个实验装置来测量附着细胞在基板上施加的凸出力。在兼容的 Formvar 板上镀的细胞变形了这种基底, 由此产生的地形被原子力显微镜 (AFM) 在纳米尺度上映射。然后从基于突出细胞结构几何的变形剖面分析中提取力值。因此, 一个活细胞的个别凸出单位施加的作用力可以随着时间的推移而测量。这一技术将使研究的力量产生和它的调节, 在许多细胞过程涉及突出。在这里, 我们描述了它的应用, 以测量由人巨噬细胞形成的 podosomes 产生的突出力。

上述协议描述了如何准备实验设置, 以量化在 Formvar 基板上应用巨噬细胞 podosomes 的突力。这是使用 AFM 实现的, 如图 1所示。
利用 JPK 数据处理软件对 podosomes 下凸起的地形图像进行分析时, 应独立地从每条扫描线中减去三度多项式拟合。可以通过在图像侧面添加 z 色刻度来评估 podosome 引起的颠簸的最大高...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

在这里, 我们详细介绍了用于评估 podosomes 在兼容薄膜上应用的突出力的实验技术, 从薄膜的制备到地形图像的自动分析。

凸出力显微镜: 一种量化细胞突起力的方法

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MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

Design and Development of AptamerÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

设计和适体 - 金纳米粒子基于比色法的开发在这领域的应用

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

单细胞阵列的活细胞成像 （LISCA） - 量化细胞动力学的多功能技术

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

使用活细胞显微镜测量扩散过程中的细胞边缘突起动力学

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...