Name: dna tension probes to map the transient piconewton receptor forces by immune cells
Uploaded: 2021-03-20T15:00:00
Description: Watch this Scientific Journal Video about DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells at JoVE.com

这项工作得到了NIH Grants R01GM131099，NIH R01GM124472和NSF CAREER 1350829的支持。我们感谢NIH四聚体设施的pMHC配体。这项研究得到了埃默里综合糖组学核心的部分支持。

OT-1转基因小鼠被饲养在埃默里大学动物资源设施部。所有实验均在机构动物护理和使用委员会（IACUC）协议下获得批准和实施。
1. 寡核苷酸制备
<ol><li>将配体链DNA溶解在水中（18.2 MΩ电阻率，在整个方案中使用）。涡旋并用台式离心机旋转溶液。调整水量，使最终浓度为1 mM。通过使用纳米滴分光光度计测量260nm处的吸光度来验证浓度，并根据寡核苷酸的消光系数确定最终浓?...

<ol>
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Y., Salaita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+nanotechnology+as+an+emerging+tool+to+study+mechanotransduction+in+living+systems.">DNA nanotechnology as an emerging tool to study mechanotransduction in living systems.</a> Small. 15 (26), 1900961 (2019).</li><li>Liu, Y., Yehl, K., Narui, Y., Salaita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+sensing+nanoparticles+for+mechano-imaging+at+the+living/nonliving+interface.">Tension sensing nanoparticles for mechano-imaging at the living/nonliving interface.</a> Journal of the American Chemical Society. 135 (14), 5320-5323 (2013).</li><li>Glazier, R., 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=DNA+mechanotechnology+reveals+that+integrin+receptors+apply+pN+forces+in+podosomes+on+fluid+substrates.">DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates.</a> Nature Communications. 10 (1), 1-13 (2019).</li><li>Galior, K., Liu, Y., Yehl, K., Vivek, S., Salaita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titin-based+nanoparticle+tension+sensors+map+high-magnitude+integrin+forces+within+focal+adhesions.">Titin-based nanoparticle tension sensors map high-magnitude integrin forces within focal adhesions.</a> Nano Letters. 16 (1), 341-348 (2016).</li><li>Zhang, Y., Ge, C., Zhu, C., Salaita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-based+digital+tension+probes+reveal+integrin+forces+during+early+cell+adhesion.">DNA-based digital tension probes reveal integrin forces during early cell adhesion.</a> Nature Communications. 5, 5167 (2014).</li><li>Liu, Y., 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=DNA-based+nanoparticle+tension+sensors+reveal+that+T-cell+receptors+transmit+defined+pN+forces+to+their+antigens+for+enhanced+fidelity.">DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.</a> Proceedings of the National Academy of Sciences. 113 (20), 5610-5615 (2016).</li><li>Zhang, Y., 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=Platelet+integrins+exhibit+anisotropic+mechanosensing+and+harness+piconewton+forces+to+mediate+platelet+aggregation.">Platelet integrins exhibit anisotropic mechanosensing and harness piconewton forces to mediate platelet aggregation.</a> Proceedings of the National Academy of Sciences. 115 (2), 325-330 (2018).</li><li>Huang, 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+kinetics+of+two-dimensional+TCR+and+pMHC+interactions+determine+T-cell+responsiveness.">The kinetics of two-dimensional TCR and pMHC interactions determine T-cell responsiveness.</a> Nature. 464 (7290), 932-936 (2010).</li><li>Ma, R., 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=DNA+probes+that+store+mechanical+information+reveal+transient+piconewton+forces+applied+by+T+cells.">DNA probes that store mechanical information reveal transient piconewton forces applied by T cells.</a> Proceedings of the National Academy of Sciences. 116 (34), 16949-16954 (2019).</li><li>Hui, E., 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=T+cell+costimulatory+receptor+CD28+is+a+primary+target+for+PD-1-mediated+inhibition.">T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.</a> Science. 355 (6332), 1428-1433 (2017).</li><li>Whitley, K. 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通过此处提供的详细程序，可以制备DNA发夹张力探针底物，以绘制和量化免疫细胞产生的受体张力。当细胞被接种到DNA发夹张力探针底物上时，当受体以化学和机械方式感知配体时，它们会着陆，附着和扩散，后者由我们的探针检测到。然而，在某些情况下，细胞可能无法扩散（图7A）或无法产生张力信号。这通常是表面化学问题的结果，需要排除故障（表1).低...

免疫细胞通过不断爬行和扫描靶细胞表面的抗原来防御病原体和癌细胞，并在其表面镶嵌1，2。抗原识别在T细胞受体（TCR）和靶细胞表面表达的肽主要组织相容性复合物MHC（pMHC）复合物结合时开始。由于TCR-pMHC识别发生在两个移动细胞之间的连接处，因此长期以来一直怀疑它经历了机械力。此外，这导致了TCR激活的机械传感器模型，这表明TCR力有助于其功能3，4。为了了解机械力何时、何地以及如何促进T细胞功能，必须开发工具来可视化T细胞传递的分子力。传统上，牵引力显微镜（TFM）和微柱阵列等方法用于研究细胞力5，6。然而，全聚焦方式（TFM）和微柱阵列的力灵敏度为纳牛顿（nN）尺度，因此通常不足以研究细胞受体传递的分子微孔（pN）力7。为了提高检测的力和空间分辨率，我们的实验室率先开发了分子张力探头，最初是使用聚乙二醇（PEG）聚合物7合成的。分子张力探针由可伸缩的分子“弹簧”（PEG、蛋白质、DNA）组成，两侧是荧光基团和淬灭剂，并固定在表面上。施加在探针末端的力导致其延伸，分离荧光团和淬灭剂，从而产生强烈的荧光信号（图1A）8，9，10。
在过去的十年中，我们开发了一个包含不同类别分子张力探针的库，其中包含由核酸11、蛋白质10 和聚合物8 制成的弹簧元件。其中，基于DNA的张力探头提供最高的信噪比和最大的力灵敏度，可在几pN到~20 pN11之间轻松调谐。我们已经使用这些实时DNA张力探针来研究许多不同细胞类型产生的分子力，包括成纤维细胞，癌细胞，血小板和免疫细胞11，12，13。本手稿将描述在表面上合成和组装DNA张力探针的方案，以使用常规荧光显微镜以pN力分辨率绘制分子受体力。虽然目前的程序包括对核酸进行化学修饰以引入荧光报告基因（图1B），但重要的是要注意，许多修饰和纯化步骤可以外包给定制DNA合成公司。因此，DNA张力探针技术是容易的，并且可以被更广泛的细胞生物学和机械生物学界所使用。
简而言之，为了组装DNA张力传感器，将DNA发夹杂交到一个臂上的荧光配体链和另一个臂上的淬灭锚链，然后固定在玻璃基板上（图1C，实时张力）。在没有机械力的情况下，发夹闭合，因此荧光被淬灭。然而，当施加的机械力大于F1/2 （平衡时的力导致50%的展开概率）时，发夹机械熔化，并产生荧光信号。
在实时DNA张力传感器的基础上，我们还描述了映射累积力的协议，这对于研究免疫细胞受体与其天然配体之间的相互作用特别有用。这是因为免疫受体通常显示短寿命的键3，14。使用“锁定”链对累积的力进行成像，该链优先结合以打开DNA发夹并允许存储与机械拉动事件相关的荧光信号（图1C，锁定张力）。锁定链旨在结合在机械诱导的发夹熔化时暴露的隐形结合位点，并通过阻止发夹重新折叠将发夹锁定在打开状态，从而存储张力信号并生成累积的张力图。此外，锁定链设计有八核苷酸链，这使得脚趾介导的链位移反应及其完整的互补，即“解锁”链。通过添加解锁链，绑定的锁定链从发夹结构上剥离，擦除存储的张力信号并将发夹重置回实时状态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62348/62348fig01.jpg"> 图 1：最先进的分子张力探头方案 。 （A）实时分子张力探针的一般设计，（B）基于DNA的张力探针构建的链，以及（C）工程化的基于DNA的张力探针及其在实时状态和锁定状态之间的切换。 <a href="https://www.jove.com/files/ftp_upload/62348/62348fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
主要方案由四个主要部分组成 - 寡核苷酸制备，表面处理，成像和数据分析。我们的实验室和其他实验室已经在幼稚和活化的OT-1 CD8 + T细胞，OT-II CD4 +细胞以及杂交瘤中成功证明了该协议，并且可以应用于询问不同的免疫细胞受体，包括T细胞受体，程序性细胞死亡受体（PD1）和淋巴细胞功能相关抗原1（LFA-1）力。本文将OT-1 CD8+幼稚T细胞用作示例细胞系。

<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;3-hydroxypicolinic acid (3-HPA)</td>
			<td>Sigma</td>
			<td>56197</td>
			<td>maldi-TOF-MS matrix</td>
		</tr>
		<tr>
			<td>&#160;mPEG-SC</td>
			<td>Biochempeg</td>
			<td>MF001023-2K</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>(3-Aminopropyl)triethoxysilane</td>
			<td>Acros</td>
			<td>AC430941000</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>10x Red blood cell lysis buffer</td>
			<td>Biolegend</td>
			<td>00-4333-57</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>8.8 nm gold nanoparticles, tannic acid</td>
			<td>Nanocomposix</td>
			<td>customized order</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>Atto647N NHS ester</td>
			<td>Sigma</td>
			<td>18373-1MG-F</td>
			<td>fluorophore, oligo prep</td>
		</tr>
		<tr>
			<td>Attofluor Cell Chamber, for microscopy</td>
			<td>Thermo Fisher Scientific</td>
			<td>A7816</td>
			<td>imaging</td>
		</tr>
		<tr>
			<td>BD Syringes only with Luer-Lok</td>
			<td>BD bioscience</td>
			<td>309657</td>
			<td>cells</td>
		</tr>
		<tr>
			<td>biotinylated anti-mouse CD3e</td>
			<td>ebioscience</td>
			<td>13-0031-82</td>
			<td>antibody/ligand</td>
		</tr>
		<tr>
			<td>Biotinylated pMHC ovalbumin (SIINFEKL)</td>
			<td>NIH Tetramer Core Facility at Emory University</td>
			<td>NA</td>
			<td>antibody/ligand</td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>735078001</td>
			<td>block non-specific interactions</td>
		</tr>
		<tr>
			<td>Cell strainers</td>
			<td>Biologix</td>
			<td>15-1100</td>
			<td>cells</td>
		</tr>
		<tr>
			<td>Coverslip Mini-Rack, teflon</td>
			<td>Thermo Fisher Scientific</td>
			<td>C14784</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>Cy3B NHS ester</td>
			<td>GE Healthcare</td>
			<td>PA63101</td>
			<td>fluorophore, oligo prep</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Sigma</td>
			<td>459836</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salts (HBSS)</td>
			<td>Sigma</td>
			<td>H8264</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>hydrogen peroxide</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>LA-PEG-SC</td>
			<td>Biochempeg</td>
			<td>HE039023-3.4K</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>Midi MACS (LS) startup kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-301</td>
			<td>cells</td>
		</tr>
		<tr>
			<td>mouse CD8+ T cell isolation kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-075</td>
			<td>cells</td>
		</tr>
		<tr>
			<td>Nanosep MF centrifugal devices</td>
			<td>Pall laboratory</td>
			<td>ODM02C35</td>
			<td>oligo prep</td>
		</tr>
		<tr>
			<td>No. 2 round glass coverslips</td>
			<td>VWR</td>
			<td>48382-085</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>NTA-SAM</td>
			<td>Dojindo Molecular Technologies</td>
			<td>N475-10</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>P2 gel</td>
			<td>Bio-rad</td>
			<td>1504118</td>
			<td>oligo prep</td>
		</tr>
		<tr>
			<td>sufuric acid</td>
			<td>EMD Millipore Corporation</td>
			<td>SX1244-6</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>Sulfo-NHS acetate</td>
			<td>Thermo Fisher Scientific</td>
			<td>26777</td>
			<td>surface prep</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent AdvanceBio Oligonucleotide C18 column, 4.6 x 150 mm, 2.7 &#956;m</td>
			<td>653950-702</td>
			<td></td>
			<td>oligonucleotide preparation</td>
		</tr>
		<tr>
			<td>Barnstead Nanopure water purifying system</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>water</td>
		</tr>
		<tr>
			<td>CFI Apo 100&#215; NA 1.49 objective</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Cy5 cube</td>
			<td>CHROMA</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>evolve electron multiplying charge coupled device (EMCCD)</td>
			<td>Photometrics</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>High-performance liquid chromatography</td>
			<td>Agilent 1100</td>
			<td></td>
			<td>oligonucleotide preparation</td>
		</tr>
		<tr>
			<td>Intensilight epifluorescence source</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS)</td>
			<td>Voyager STR</td>
			<td></td>
			<td>oligonucleotide preparation</td>
		</tr>
		<tr>
			<td>Nanodrop 2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>oligonucleotide preparation</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti inverted microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Nikon Perfect Focus System</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>quad band TIRF 405/488/561/647 cube</td>
			<td>CHROMA</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>RICM cube</td>
			<td>CHROMA</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>TIRF launcher with 488 nm (50 mW), 561 nm (50 mW), and 640 nm</td>
			<td>Coherent</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>TRITC cube</td>
			<td>CHROMA</td>
			<td></td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>oligo name</td>
			<td>5&#39; modification / 3&#39; modification</td>
			<td>sequence (5&#39; to 3&#39;)</td>
			<td>Use</td>
		</tr>
		<tr>
			<td>15mer amine locking strand</td>
			<td>5&#39; modification: no modification 
			3&#39; modification: /3AmMO/</td>
			<td>AAA AAA CAT TTA TAC CCT ACC TA</td>
			<td>locking real-time tension signal</td>
		</tr>
		<tr>
			<td>15mer Atto647N locking strand</td>
			<td>5&#39; modification: Atto647N 
			3&#39; modification: /3AmMO/</td>
			<td>AAA AAA CAT TTA TAC CCT ACC TA</td>
			<td>locking real-time tension signal</td>
		</tr>
		<tr>
			<td>15mer non-fluoresccent locking strand</td>
			<td>5&#39; modification: no modification 
			3&#39; modification: no modification</td>
			<td>A AAA AAC ATT TAT AC</td>
			<td>locking real-time tension signal for quantitative analysis</td>
		</tr>
		<tr>
			<td>4.7 pN hairpin strand</td>
			<td>5&#39; modification: no modification 
			3&#39; modification: no modification</td>
			<td>GTGAAATACCGCACAGATGCGT 
 TTGTATAAATGTTTTTTTCATTTAT 
 ACTTTAAGAGCGCCACGTAGCC 
CAGC</td>
			<td>hairpin probe</td>
		</tr>
		<tr>
			<td>amine ligand strand</td>
			<td>5&#39; modification: /5AmMC6/ 
			3&#39; modification: /3Bio/</td>
			<td>CGCATCTGTGCG GTA TTT CAC TTT</td>
			<td>hairpin probe</td>
		</tr>
		<tr>
			<td>BHQ2 anchor strand</td>
			<td>5&#39; modification: /5ThiolMC6-D/ 
			3&#39; modification: /3BHQ_2/</td>
			<td>TTTGCTGGGCTACGTGGCGCTCTT</td>
			<td>&#160;&#160;&#160;hairpin probe</td>
		</tr>
		<tr>
			<td>Cy3B ligand strand</td>
			<td>5&#39; modification: Cy3B 
			3&#39; modification: /3Bio/</td>
			<td>CGCATCTGTGCG GTA TTT CAC TTT</td>
			<td>hairpin probe</td>
		</tr>
		<tr>
			<td>unlocking strand</td>
			<td>5&#39; modification: no modification 
			3&#39; modification: no modification</td>
			<td>TAG GTA GGG TAT AAA TGT TTT TTT C</td>
			<td>unlocking accumulated tension signal</td>
		</tr>
	</tbody>
</table>

dna tension probes to map the transient piconewton receptor forces by immune cells

在两个相邻细胞之间的连接处以及细胞与细胞外基质之间的连接处传递的机械力对于调节从发育到免疫学的许多过程至关重要。因此，开发在分子尺度上研究这些力的工具至关重要。我们小组开发了一套分子张力传感器，用于量化和可视化细胞产生并传递到特定配体的力。最灵敏的一类分子张力传感器由核酸茎环发夹组成。这些传感器使用荧光团-淬灭剂对来报告DNA发夹在力下的机械延伸和展开。DNA发夹张力传感器的一个挑战是，它们是可逆的，在张力终止时快速重新折叠发夹，因此很难记录瞬态力。在本文中，我们描述了制备DNA张力传感器的协议，这些传感器可以“锁定”并防止重新折叠，以实现机械信息的“存储”。这允许记录高瞬态微微顿力，随后可以通过添加去除锁的互补核酸来“擦除”。这种在实时张力映射和机械信息存储之间切换的能力揭示了弱的、短暂的和不太丰富的力，这些力通常被T细胞用作其免疫功能的一部分。

在这里，我们显示了具有代表性的表面质量控制图像（图4）。高质量的表面应在RICM通道中具有干净的背景（图4B），在Cy3B通道中应具有均匀的荧光强度（图4C）。在相同的成像设备和相同的荧光成像采集条件下，在使用DNA探针进行实验时，每次背景荧光强度应一致且可重现。我们建议在每组单独实验之前将其用作表面质量控制的...

Watch this Scientific Journal Video about DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells at JoVE.com

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

本文描述了使用基于DNA的张力探针对免疫细胞施加的受体力进行成像的详细方案。这种方法可以实时映射受体力>4.7pN，并且可以随着时间的推移积分力。

DNA张力探针用于绘制免疫细胞的瞬时皮牛顿受体力

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

该协议提供了一种绘制细胞，特别是免疫细胞产生的分子力的方法。该协议对于有兴趣研究机械生物学机械转导的实验室至关重要。该技术的主要优点是它非常容易，并且允许使用传统的荧光显微镜来量化永生化细胞和原代细胞中的力传递。首先，将 25 毫米盖玻片放在 50 毫升烧杯中的聚四氟乙烯架上，并通过将它们浸入水中来冲洗盖玻片三次。混合等体积的乙醇和水，并将40毫升该溶液加入装有架子和盖玻片的烧杯中。然后，使用封口胶带密封烧杯。通过在超声波清洁器中超声处理烧杯 15 分钟来清洁盖玻片，并在超声处理后丢弃液体。然后，用水冲洗装有架子和盖玻片的烧杯至少六次，以去除任何残留的有机溶剂。要制备新鲜的食人鱼溶液，请在 50 毫升烧杯中加入 30 毫升硫酸，然后慢慢加入 10 毫升过氧化氢。使用玻璃移液管的末端轻轻混合食人鱼溶液。要开始蚀刻，将装有盖玻片的架子转移到装有食人鱼溶液的烧杯中，并在室温下孵育 30 分钟以清洁盖玻片并将其羟基化。然后，使用钢或聚四氟乙烯镊子将架子转移到干净的 50 毫升烧杯中，并用水冲洗六次。为了完全除去水，将装有盖玻片的架子浸入装有40毫升乙醇的50毫升烧杯中。然后，丢弃乙醇。接下来，将架子浸入含有3%氨丙基三乙氧基硅烷的40毫升乙醇中，在室温下一小时，以引发与盖玻片上的羟基的反应。将盖玻片浸入40毫升乙醇中冲洗盖玻片表面六次，然后在80摄氏度的烤箱中干燥20分钟。用封口膜胶带覆盖培养皿的内底部，以防止盖玻片在培养皿内滑动。然后将胺覆盖的盖玻片放在要功能化的一侧，DNA张力探针朝上。盖上盖玻片，温度为20摄氏度，以备将来使用。要修饰盖玻片上的胺基团，加入硫辛酸PEG NHS和mPEG NHS，并将培养皿在室温下孵育一小时。在孵育结束时，用水冲洗表面三次。之后，将100微升0.1摩尔碳酸氢钠加入一组夹心盖玻片中，每毫升含有1毫克磺基-NHS-乙酸酯，并孵育30分钟以进行钝化。向每个盖玻片中加入0.5毫升金纳米颗粒，并在室温下孵育30分钟。孵育后，用水冲洗盖玻片三次。将黑洞淬灭2链以10比1的比例加入退火的DNA张力探针后，每两个盖玻片加入100微升混合物以制作三明治。小心地将湿的实验室组织球放在远离盖玻片的培养皿中，并用封口膜胶带密封培养皿以防止溶液干燥。之后，用箔纸盖住培养皿，并在四摄氏度下孵育过夜。第二天，组装成像室，在拧紧成像室时注意防止表面开裂。然后，向成像室中加入0.5至1毫升汉克平衡盐溶液。使用带有100倍物镜的Cy3B通道，在DNA发夹张力探针开始扩散时捕获接种在DNA发夹张力探针上的细胞的荧光信号。制备100微摩尔非荧光锁定链储备液后，在所需时间点将其添加到成像室中终浓度为一微摩尔的细胞中以存储张力信号并通过移液将其与细胞混合。锁定约10分钟后，获取落射荧光中的延时动画或终点图像，以进行定性张力映射和定量分析。成功的表面呈淡粉红色。在干净的背景和反射干涉对比显微镜或RICM通道以及Cy3B通道中均匀的荧光强度下，证明了表面的高质量。张力锁定的延时图像表明，由于发夹锁定和信号积累，TCR抗CD3ε和TCR-肽-MHC张力信号均被放大。锁定链记录了从零分钟开始的TCR肽MHCN4的短暂机械拉扯事件，这有助于定量分析，并揭示了类似于免疫突触靶心模式的TCR肽MHC力的独特模式。每一步，您的意向探针基板制备都需要注意细节并小心处理材料。该技术能够可视化细胞表面与环境相互作用时发生的瞬态分子力，特别是在寻找抗原的免疫细胞中。

Research

JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

在激光诱导轴突病变张力释放测量评估轴突在Piconewton和毫秒级的分辨率对基材的附着力

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

柔性神经微探针的使用附加与Biodissolvable胶粘剂刚性加劲肋插入

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

的剪切粘合地图使用SynVivo合成微血管网络代

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging

基于 CMUT 技术的新型超声探头在脑成像中的应用研究

The possibility to perform an early and repeatable assessment of imaging performance is fundamental in the design and development process of new ...

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis

早期雏鸡胚胎形态发生中物理力的作用探讨

Embryonic development is traditionally studied from the perspective of biomolecular genetics, but the fundamental importance of mechanics in morphogenesis ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

集成张力传感器在亚微米分辨率下的成像整合和细胞力

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...