Name: functionalization of atomic force microscope cantilevers with single-t cells or single-particle for immunological single-cell force spectroscopy
Uploaded: 2019-07-10T13:30:00
Description: Watch this Scientific Journal Video about Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy at JoVE.com

这项工作得到了国家自然科学基金总体计划(31370878)、国家重点计划(31630023)和创新研究组计划(81621002)的支持。

小鼠实验方案遵循清华大学动物护理指南
1. 单T细胞的悬臂功能化
<ol><li>小鼠脾细胞制剂<ol>
<li>牺牲小鼠(8-16周的年龄(两性);例如,C57BL/6菌株)使用二氧化碳,其次是宫颈脱位。</li>
		<li>用75%乙醇清洁小鼠,进行中线皮肤切口,然后进行切除。</li>
		<li>使用玻璃滑梯将含有 2% 胎儿牛血清 (FBS) 的 PBS 4 mL 中的脾脏均匀化,并通过 70 μm 网状尼龙滤网将细胞悬浮液排出骨质和碎屑。</li>
		<...

<ol>
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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=Binding+strength+and+dynamics+of+invariant+natural+killer+cell+T+cell+receptor/CD1d-glycosphingolipid+interaction+on+living+cells+by+single+molecule+force+spectroscopy.">Binding strength and dynamics of invariant natural killer cell T cell receptor/CD1d-glycosphingolipid interaction on living cells by single molecule force spectroscopy.</a> Journal of Biological Chemistry. 286 (18), 15973-15979 (2011).</li><li>Flach, T. 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=Alum+interaction+with+dendritic+cell+membrane+lipids+is+essential+for+its+adjuvanticity.">Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity.</a> Nature Medicine. 17 (4), 479-487 (2011).</li><li>Ng, G., 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=Receptor-independent,+direct+membrane+binding+leads+to+cell-surface+lipid+sorting+and+Syk+kinase+activation+in+dendritic+cells.">Receptor-independent, direct membrane binding leads to cell-surface lipid sorting and Syk kinase activation in dendritic cells.</a> Immunity. 29 (5), 807-818 (2008).</li><li>Helenius, J., Heisenberg, C. P., Gaub, H. E., Muller, D. 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T., Horton, 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=Single+cell+mechanotransduction+and+its+modulation+analyzed+by+atomic+force+microscope+indentation.">Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation.</a> Biophysics Journal. 82 (6), 2970-2981 (2002).</li><li>Sun, M. Z., 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=Multiple+membrane+tethers+probed+by+atomic+force+microscopy.">Multiple membrane tethers probed by atomic force microscopy.</a> Biophysics Journal. 89 (6), 4320-4329 (2005).</li><li>Yan, J. C., Liu, B., Shi, Y., Qi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class+II+MHC-independent+suppressive+adhesion+of+dendritic+cells+by+regulatory+T+cells+in+vivo.">Class II MHC-independent suppressive adhesion of dendritic cells by regulatory T cells in vivo.</a> Journal of Experimental Medicine. 214 (2), 319-326 (2017).</li><li>Hao, J. 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=Phospholipase+C-mediated+hydrolysis+of+PIP2+releases+ERM+proteins+from+lymphocyte+membrane.">Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane.</a> Journal of Cell Biology. 184 (3), 451-462 (2009).</li><li>Rodriguez, R. 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=Lymphocyte-T+Adhesion+to+Fibronectin+(Fn)+-+a+Possible+Mechanism+for+T-Cell+Accumulation+in+the+Rheumatoid+Joint.">Lymphocyte-T Adhesion to Fibronectin (Fn) - a Possible Mechanism for T-Cell Accumulation in the Rheumatoid Joint.</a> Clinical and Experimental Immunology. 89 (3), 439-445 (1992).</li><li>Kimura, A., Ersson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Lymphocytes-T+by+Lectins+and+Carbohydrate-Oxidizing+Reagents+Viewed+as+an+Immunological+Recognition+of+Cell-Surface+Modifications+Seen+in+the+Context+of+Self+Major+Histocompatibility+Complex+Antigens.">Activation of Lymphocytes-T by Lectins and Carbohydrate-Oxidizing Reagents Viewed as an Immunological Recognition of Cell-Surface Modifications Seen in the Context of Self Major Histocompatibility Complex Antigens.</a> European Journal of Immunology. 11 (6), 475-483 (1981).</li><li>Miller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stimulation+of+Human+Lymphocyte-B+and+Lymphocyte-T+by+Various+Lectins.">The Stimulation of Human Lymphocyte-B and Lymphocyte-T by Various Lectins.</a> Immunobiology. 165 (2), 132-146 (1983).</li><li>Vitte, J., Pierres, A., Benoliel, A. 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基于AFM的单细胞力光谱已经演变为解决活细胞生物物理特性的有力工具。对于这些应用,悬臂需要正确工作,以便探测感兴趣的单元上的特定交互或属性。分别介绍了单T单元和单微米尺寸珠与无尖悬臂耦合的方法。为了将单个T细胞附着在悬臂上,选择生物相容性胶水作为细胞粘合剂。它是一种从海洋贝类中提取的特制蛋白质溶液。其粘附性源于多酚残留物,其羟基基组可与以非特异性方式在细胞表面?...

原子力显微镜(AFM),一个多功能的工具,在细胞生物学研究1,2,3,4,5中已经发现许多应用。除了高分辨率成像能力外,原生力探测功能还允许活细胞的生物物理特性在单细胞6、7级直接就地研究。这些包括亚细胞结构,甚至整个细胞的刚性 8,9,10,11,12,特定的配体/受体结合强度细胞表面的单分子水平13,固体粒子和细胞的单对之间的附着力或两个细胞1、2、14、15之间的附着力。后两种通常被归类为单细胞力光谱(SCFS)16。由于各种弹簧常数的易得的悬臂,AFM 可访问的力范围相当广泛,从几皮克纽顿 (pN) 到微牛顿 (μN),这足以覆盖涉及几十个力的整个细胞事件范围pN,如基于受体的单分子结合,到nN,如噬菌体细胞事件15。这种较大的动态力范围使 AFM 优于其他力探测技术,如光学/磁性钳子和生物膜力探头,因为它们更适合弱力测量,其力通常小于 200 pN17,18.此外,AFM可以作为高精度操纵器,以临时定义的方式4,19向单个细胞提供各种刺激。这是理想的实时单细胞活化研究。结合活细胞荧光成像,可以同时监测随后细胞对特定刺激的反应,从而使基于AFM的SCFS作为光学成像极其强大,为探测细胞信号提供了实用的工具。例如,AFM用于确定在成骨菌20中引起钙瞬变所需的菌株。在这项工作中,在用AFM尖端在培养的成骨器上应用局部力后,通过钙比例成像对钙瞬变进行荧光跟踪。最近,AFM被用于拉伸胶原纤维,在胶原纤维上生长肝硬质细胞(HSC),这种经研究用的HSC活化由荧光Src生物传感器实时监测,其磷酸化代表生物传感器的荧光强度与HSC活化3相关。
在基于 AFM 的 SCFS 实验中,AFM 悬臂的正确功能化是成功测量的关键步骤。由于我们的研究兴趣集中在免疫细胞激活,我们经常功能与颗粒物质,如单个固体颗粒,可以触发噬菌体和/或强烈的免疫反应4,14,15和单个T细胞,可以形成免疫突触与抗原呈现细胞,如活化树突细胞(DC)2。单固体颗粒通常通过空气环境中的环氧胶与悬臂耦合,而单 T 细胞由于其非粘合性质,通过溶液中的生物相容性胶水功能化为悬臂。在这里,我们将描述执行这两种类型的悬臂修改的方法,并给出两个关联的应用程序。第一个应用是探测T细胞/直流与AFM-SCFS的相互作用,以便从细胞力学的角度理解调节T细胞的抑制机制。第二个涉及将AFM与活细胞荧光成像相结合,实时监测巨噬细胞对固体颗粒的细胞反应,揭示受体独立磷脂醇4,5-双磷酸(PIP2)的分子机制-莫辛调解了方位体。该协议的目的是为感兴趣的研究人员提供一个参考框架,以设计和实现自己的实验设置与基于AFM的单细胞分析的免疫学研究。

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;l pipette tip</td>
			<td>Thermo Fisher</td>
			<td>104-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>6 cm diameter culture dish</td>
			<td>NALGENE nunc</td>
			<td>150462</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>JET</td>
			<td>TCP011006</td>
			<td></td>
		</tr>
		<tr>
			<td>AFM Cantilever</td>
			<td>NanoWorld</td>
			<td>Arrow-TL1-50</td>
			<td>tipless cantilever</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>7604</td>
			<td></td>
		</tr>
		<tr>
			<td>Biocompatible glue</td>
			<td>BD Cell-Tak</td>
			<td>354240</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4+ T cell isolation Cocktail</td>
			<td>STEMCELL</td>
			<td>19852C.1</td>
			<td></td>
		</tr>
		<tr>
			<td>DC2.4 cell line</td>
			<td>A gift from K. Rock (University of Massachusetts Medical School, Worcester, MA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran-coated magnetic particles</td>
			<td>STEMCELL</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>GENEray</td>
			<td>Generay-E1101-500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>ERGO</td>
			<td>7100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>twbio</td>
			<td>00019</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Ex Cell Bio</td>
			<td>FSP500</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR blocker</td>
			<td>STEMCELL</td>
			<td>18731</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>local vender (Hai Men Lian Sheng)</td>
			<td>HX-E37</td>
			<td>24mm diameter, 0.17mm thinckness</td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>JinTong department of laboratory and equipment management, Haimen&#160;</td>
			<td>N/A</td>
			<td>customized</td>
		</tr>
		<tr>
			<td>H2O2 (30%)</td>
			<td>Sino pharm</td>
			<td>10011218</td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sino pharm</td>
			<td>80120892</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>51558</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>STEMCELL</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh nylon strainer</td>
			<td>BD Falcon</td>
			<td>REF 352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Moesin-EGFP</td>
			<td>N/A</td>
			<td></td>
			<td>cloned in laboratory</td>
		</tr>
		<tr>
			<td>Mouse CD25 Treg cell positive isolation kit</td>
			<td>STEMCELL</td>
			<td>18782</td>
			<td>Component: FcR Blocker,Regulatory T cell Positive Selection Cocktail, PE Selection Cocktail, Dextran RapidSpheres,</td>
		</tr>
		<tr>
			<td>Mouse CD4+ Tcell isolation kit</td>
			<td>STEMCELL</td>
			<td>19852</td>
			<td>Component:CD4+T cell isolation Cocktail, Streptavidin RapidSpheres, Rat Serum</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Lanyi chemical products co., LTD&#65292; Beijing</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1022-500</td>
			<td></td>
		</tr>
		<tr>
			<td>PE selection cocktail</td>
			<td>STEMCELL</td>
			<td>18151</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>PLC&#948;-PH-mCherry</td>
			<td>Addgene</td>
			<td>36075</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres 6.0&#956;m</td>
			<td>Polysciences</td>
			<td>07312-5</td>
			<td></td>
		</tr>
		<tr>
			<td>polystyrene round bottom tube</td>
			<td>BD Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>STEMCELL</td>
			<td>13551</td>
			<td></td>
		</tr>
		<tr>
			<td>RAW264.7</td>
			<td>&#160;ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-2</td>
			<td>Peprotech</td>
			<td>Peprotech, 200-02-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td></td>
		</tr>
		<tr>
			<td>Regulatory T cell positive selection cocktail</td>
			<td>STEMCELL</td>
			<td>18782C</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life</td>
			<td>C11875500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-coated magnetic particles</td>
			<td>STEMCELL</td>
			<td>50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection kit</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% EDTA</td>
			<td>Life</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>JD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x objective NA 0.8</td>
			<td>Zeiss</td>
			<td>420650-9901</td>
			<td>Plan-Apochromat</td>
		</tr>
		<tr>
			<td>Atomic force microscope</td>
			<td>JPK</td>
			<td>cellHesion200</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman coulter</td>
			<td>Allegra X-12R</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence imaging</td>
			<td>home-made objective-type total internal reflection fluorescence microscop based on a Zeiss microscope stand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>HERACELL 150i</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Zeiss</td>
			<td>Observer A1 manual</td>
			<td></td>
		</tr>
	</tbody>
</table>

functionalization of atomic force microscope cantilevers with single-t cells or single-particle for immunological single-cell force spectroscopy

基于原子力显微镜的单细胞力光谱(AFM-SCFS)是研究活细胞生物物理特性的有力工具。这种技术允许在活细胞膜上探测相互作用强度和动力学,包括细胞、受体和配体之间的相互作用强度和动力学,以及许多其他变异。它还作为一种机制,以时空控制的方式在单个细胞上提供物理或生化刺激,从而在与活细胞结合时实时监控特定的细胞激活和随后的细胞事件荧光成像。这些 AFM-SCFS 测量的关键步骤是 AFM 悬臂功能化,换句话说,将感兴趣的主题附加到悬臂上。在这里,我们提出用单个T细胞和单个聚苯乙烯珠分别修改AFM吊杆的方法,用于免疫学研究。前者涉及一种生物相容性胶水,将单T细胞耦合到溶液中的扁平悬臂尖端,而后者则依靠环氧胶在空气环境中的单珠粘附。还提供了与每个悬臂修饰相关的两个免疫学应用。此处描述的方法可以很容易地适应不同的细胞类型和固体颗粒。

图 4A显示了单 T 单元和单 DC 之间在一个接近缩回周期中结合交互的典型力距离曲线。浅红色曲线是延伸曲线,深红色曲线为回缩曲线。由于延伸曲线通常用于缩进或刚度分析,因此这里只涉及缩进曲线, 与细胞粘附有关。曲线中的最小值(绿色圆圈)表示最大附着力的度量。曲线下的区域(带沙区)表示将 T 单元与 DC 分离所需的工作(能量)。在完全分离之前,...

Watch this Scientific Journal Video about Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy at JoVE.com

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

我们提出了一种协议,用单个T细胞和珠子粒子使原子力显微镜(AFM)的吊带功能化,用于免疫学研究。显示了通过AFM探测单对T细胞树突状细胞结合的过程,并监测宏噬细胞对单个固体颗粒的实时细胞响应,并采用荧光成像。

原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

该协议描述了如何使用单个 T 细胞和固体粒子以最佳方式使 AFM 推进体功能化。然后，这些提示可用于探测单对树突状细胞-T细胞相互作用，并分别监测变化无常大小的细胞反应。该技术的主要优点是，它使用生物相容性胶水进行单T细胞功能化的可逆剂。这种胶水对真核细胞是惰性的，从而使T细胞处于基线激活阶段。这些方法提供对免疫细胞活化和复杂的细胞间事件，如免疫突触形成的洞察。这些方法也可以扩展到涉及细胞细胞或细胞表面相互作用的其他系统。对于对这项技术很了解的人，重要的是确保T细胞在使用前处于良好状态，并且一夜之间用IL-2进行预处理。此外，使用生物相容性胶水时，请快速移动，防止不必要的氧化。要开始此过程，请按照文本协议中遵循，为显微镜准备单个 T 细胞。然后，准备用DC2.4细胞播种的玻璃盖玻片，在37摄氏度和5%CO2的加湿室中孵育细胞过夜。使用食人鱼法处理、血浆清洁或紫外线臭氧清洁，清洁柔软、无尖的软性针干剂，具有低弹簧常数。然后，将清洁的悬臂安装到 AFM 扫描头上。接下来，准备一个干净的样品室，并用纯净水填充。首先在玻璃基板上运行力曲线，以获得悬臂的灵敏度，校准水溶液中的悬臂。然后，记录热噪声频谱以提取弹簧常数。正确校准悬臂后，从溶液中卸下 AFM 扫描头。用几滴纯乙醇清洗安装的悬臂，使悬臂在扫描头上保持干燥。将活细胞环境外壳预热至 37 摄氏度，二氧化碳为 5%。然后，将用 DC2.4 电池播种的玻璃盖玻片安装到样品室组件中，并立即向样品室中加入 600 微升的中B。将已完成的组件放在 AFM 样品阶段。将人类IL-2孵育CD4阳性T细胞加入样品室。等待目标T细胞完全安放在盖玻片底部，然后在显微镜下将细胞放入视野。现在，用移液器将两微升的生物相容性胶水滴到安装的悬臂的末端。一旦生物相容性胶水被应用到悬臂上，需要尽快完成后续步骤，以最大限度地提高粘附性。快速将扫描头放在样品阶段，将涂胶的悬臂浸入溶液中。移动样品级，直到位于悬臂尖端下方的运行状况 T 细胞。然后，通过移动扫描头微调其位置。接下来，手动降低悬臂。从 50 微米步进开始，然后逐渐减小到 10、5 和 2，最后为 0.5 微米，如悬臂图像的锐度所示。现在，按住步进电机的位置，并调整扫描头的位置，以更好地对齐悬臂尖端和电池。继续，直到激光位置的小位移指示牢固接触，对应于 0.5 至 1.5 纳米新吨的典型力。接触30秒后，收回悬臂。如果单元格随悬臂移动，则附件成功。如果没有，在切换到新的悬臂之前，重复粘附步长达三次。通过移动样品级和扫描头，将附加的 T 单元放在 DC2.4 单元上方。设置适当的参数后，对样品进行力光谱分析。对于新一轮的探测，安装一个新的，干净的悬臂。在纯水中校准它，然后返回T细胞-树突状细胞对并重复扫描。将清洁的玻璃盖玻片安装到样品室组件上。在盖玻片的左侧，添加一滴在乙醇中稀释并含有六微米直径聚苯乙烯珠的珠子溶液。溶剂蒸发后，使用配备 20 倍目标的亮场显微镜检查珠子的间距。确保各个珠子分离良好。然后，将微管尖端或牙签浸入混合良好的环氧树脂胶中，将少量胶水转移到三个单独的点中，右侧连续轻轻接触，但靠近盖玻片的中心，如下所示。接下来，将清洁的无尖悬臂安装到 AFM 扫描头上，并在空气中用干净的表面校准，以获得弹簧常数。然后，将悬臂笔尖放在最后一个环氧胶水点左边界上，然后用步进电机上的小步进尺寸慢慢将悬臂靠近胶水。与胶水接触后，通过向后移动 AFM 扫描头，快速横向拉拔悬臂。通过擦掉玻璃上的任何多余的胶水，在尖端的末尾只留下少量胶水。现在，将悬臂尖端移在隔离良好的珠子上。慢慢接近单珠，在2到5纳米新珠的范围内与它进行坚定的接触，约10秒。进行接触时，使用细尖调整将珠子定位在尖端的末尾。缩回触点端的尖端。如果珠子从原始焦点平面上消失，则发生成功的粘附事件。最后，小心地将珠子修改的悬臂卸下，并将其存放在悬臂盒中过夜，使环氧树脂完全固化。以下是单个 T 单元和单个树突状细胞在一个方法缩回循环过程中结合相互作用的典型力-距离曲线。浅红色曲线是延伸曲线，深红色曲线是缩回曲线。曲线中的最小值表示 T 细胞和树突状细胞之间的最大粘附力，曲线下的面积表示分离它们所需的工作。尖锐和逐步的破裂事件可以解释为膜系绳被从细胞表面拉出，由于在细胞接口的强结合，然后在连续拉下离散断裂。在这里，常规T细胞与树突状细胞结合以灰色显示，与树突状细胞结合的调节T细胞以蓝色显示。常规T细胞和调节T细胞之间的粘附力可识别抗原呈现细胞呈现的肽抗原，而调节T细胞是抑制剂T细胞，在免疫反应接近尾声时关闭传统的T细胞调节的免疫力。这个荧光标记的噬菌体 RAW264.7 细胞的示意图显示细胞正在接近一个赤裸的、直径为 6 微米的聚苯乙烯珠。接触珠子后，膜 PIP2 在接触点（靠近 b）进行排序，然后诱导膜招募mosin，从而引发噬菌论事件。除了此处所述的程序外，基于 AFM 的单细胞力光谱学可与荧光成像一起使用，在单细胞水平上实时研究免疫细胞激活。结合荧光技术，此方法允许实时处理更复杂的细胞过程，例如在树突状细胞和T细胞对之间形成免疫突触。

Research

JoVE Journal

Immunology and Infection

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

促进免疫学数据分析与视觉分析技术

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

科研

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

诱导细胞分化和单细胞成像<em&gt;副溶血性弧菌</em游泳者和Swarmer细胞

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...