我们感谢 Nurul Aisha Zainal Abidin 和 Laura Moldovan 为献血者招募、采血和放血提供额外支持。我们感谢托马斯·安德森（Tomas Anderson）和阿里安·纳赛尔（Arian Nasser）组织设备和试剂。这项研究由澳大利亚研究委员会（ARC）发现项目（DP200101970-L）资助。A.J.）;澳大利亚国家健康与医学研究委员会 （NHMRC） 创意资助 （APP2003904-L.A.J.）;NHMRC 设备补助金-LAJ;新南威尔士州心血管能力建设计划（Early-Mid Career Researcher Grant-LAJ）;新南威尔士州CVRN-VCCRI研究创新补助金;全球和研究参与办公室（悉尼-格拉斯哥伙伴关系合作奖-LAJ）;L.A.J. 是美国国家心脏基金会未来领袖研究员 2 级 （105863） 和斯诺医学研究基金会研究员 （2022SF176）。

该协议遵循悉尼大学人类研究伦理委员会的指南并已获得批准。本研究已获得捐赠者的知情同意。
1. 人红细胞分离
注意：步骤 1.1 应由训练有素的抽血师使用机构审查委员会批准的方案执行。
<ol><li>使用 19 G 蝴蝶针从肘正中静脉抽取 5 mL 血液。</li>
	<li>将收集的血液转移到含有 1：200 依诺肝素的 15 mL 试管中以防止凝血。</li>
	<li>在 1 mL 碳酸盐/碳酸氢盐缓冲液（C 缓冲液，pH = 8.5-9; 材料表）。</li>
	<li>将稀释的血液样品以900× g 离心1分钟以沉淀红细胞。 小心地倾析上清液而不干扰沉淀。</li>
	<li>用 1 mL C 缓冲液（材料表）洗涤 RBC 沉淀两次，每次以 900 × g 离心 1 分钟。</li>
	<li>随后，使用相同的离心条件用 1 mL Tyrode 缓冲液洗涤 RBC 沉淀 2 次，然后将最终沉淀重悬于 1 mL Tyrode 缓冲液中以获得洗涤后的原液 RBC 悬浮液。</li>
</ol>2.钙指示剂负荷
<ol><li>根据使用自动细胞计数器（材料表）获得的细胞计数，将洗涤原液 RBC 溶液的浓度在 Tyrode 缓冲液中调节至 10 ×10 6 个细胞/mL。</li>
	<li>通过与16.67μM Cal-520AM（一种钙敏感染料）一起孵育，同时在旋转管混合器上搅拌1小时，标记RBC内的钙。</li>
	<li>在含有 0.5% 牛血清白蛋白 （BSA） 的 Tyrode 缓冲液中以 1：50 的比例稀释红细胞。这些细胞现在已准备好用于实验。</li>
</ol>3. 微量移液器制造
<ol><li>将硼硅酸盐玻璃毛细管（外径 1 mm x 内径 0.6 mm）安装到 P-1000 微量移液器拉拔器上，以使用预设的拉拔程序在拉拔部位产生两个相应的带有闭塞吸头的微量移液器。对于此设置，请使用以下拉动程序值：热量 516、拉力 150、速度 75、时间 250 和 压力 500。 注：在拉动程序中设置的加热和拉动参数可以定制，并且取决于实验设计12的所需设置。 CHECKPOINT （参见 补充表 S1）。</li>
	<li>通过安装拉到微量移液器切割器上后获得的封闭式微量移液器之一来打开封闭的吸头。将加热温度调节至约50-60°C。</li>
	<li>使用10倍目镜定位微量移液器。使用旋钮进行调整，将微量移液器靠近硼硅酸盐玻璃珠。</li>
	<li>将目镜更换至 30 倍，然后将微量移液器放置在尽可能靠近硼硅酸盐玻璃珠的位置，而无需弯曲移液器尖端。</li>
	<li>踩在加热踏板上，用热量软化硼硅酸盐玻璃珠。轻轻地将未密封的微量移液器吸头插入软化的微珠中，直到达到所需的终点，即开口直径。</li>
	<li>松开脚踏板，让玻璃珠冷却下来。确保微量移液器的尖端始终留在珠子内。 注意： 进一步插入尖端会导致更大的开口直径。</li>
	<li>轻轻抽出微量移液器，在封闭的微量移液器上形成清晰的直切口。确认毛细管的最终直径为1μm。 注： CHECKPOINT （参见 补充表 S1）</li>
</ol>4. 细胞室制备
<ol><li>使用金刚石铅笔将标准的 40 mm x 22 mm x 0.17 mm 玻璃盖玻片分成三个相等的条带。</li>
	<li>用真空润滑脂将一块切割好的玻璃盖玻片粘贴到自制腔室支架的底部。 注意： 腔室支架由两个金属（铜/铝）正方形组成，它们通过弯曲的手柄连接。金属块之间的距离必须小于 40 毫米，切割的盖玻片才能粘附在支架上以形成平行腔室。</li>
	<li>用真空润滑脂将切割玻璃盖玻片的第二块粘附在自制腔室支架的顶部。 注： CHECKPOINT （参见 补充表 S1）</li>
	<li>使用 200 μL 移液管枪在两个盖玻片之间注入 200 μL 标记的 RBC 悬浮液（图 1）。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66265/66265fig01.jpg"> 图 1：细胞室的图示。 使用润滑脂将两块 40 mm x 22 mm x 0.17 mm 玻璃盖玻片的切割件粘附在腔室支架上。在两个切割的玻璃盖玻片之间，接种约200μLTyrode缓冲液中的细胞溶液。 <a href="https://www.jove.com/files/ftp_upload/66265/66265fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
5. 微量移液器抽吸组件
<ol><li>将细胞室安装到显微镜平台上的支架上。调整位置，使细胞室位于物镜正上方（图2B）。</li>
	<li>将微量移液器支架降低到所连接储水器的液位以下。</li>
	<li>将软化水或Tyrode缓冲液注入制备的微量移液器中，并使用带有34G针头的注射器小心地除去所有气泡（参见材料表）。</li>
	<li>将微量移液器支架的末端拧下一半，让水从微量移液器支架上滴落几秒钟。 注： CHECKPOINT （参见 补充表 S1）</li>
	<li>将微量移液器插入支架吸头。拧紧支架螺钉，确保微量移液器固定。</li>
	<li>将微量移液器插入细胞室，并在显微镜下定位微量移液器和红细胞。使用显微操纵器调整位置。</li>
	<li>进一步降低微量移液器吸头，以确保吸头与位于的红细胞齐平。 注： CHECKPOINT （参见 补充表 S1）</li>
	<li>通过调节储水器的高度，将微量移液器吸头处的液压归零。然后，稍微抬高储水器，在尖端产生微妙的正压。</li>
</ol>6. 进行荧光偶联微量移液器抽吸测定
<ol><li>打开 488 nm 荧光激发光源。在此阶段不要打开荧光快门以避免光漂白（图2C）。打开荧光相机和透射相机。 注意： 两台相机均使用适当的软件进行操作（参见 材料表）。</li>
	<li>在软件中为两台相机设置所需的 曝光时间 （本研究中两台相机均为 100 毫秒 ）、感兴趣区域 （ROI）、像素合并大小（本研究中无）。打开 多维采集面板 ，设置 采集帧号，本研究2000个，保存目录。 注意：采集帧数取决于要记录的所需抽吸事件数量。对于 1 次抽吸事件，采集数的范围应设置在 100-500 以内，约为 10-50 s。</li>
	<li>使用显微操纵器在视野下找到微量移液器。</li>
	<li>打开气动压力夹，包括控制箱和夹具系统（图 2A）。确保 控制箱 处于 EXTRNL 模式。通过缓慢旋转旋钮来补偿系统内的任何偏移压力。</li>
	<li>打开控制气动夹的单独软件amp。该软件具有一个电气控制面板，用于控制钳位系统的离散模拟输入。压力控制在20 mV/mmHg转换系数上。</li>
	<li>将系统内部的压力归零。小心地将微量移液器靠近红细胞的位置。 调整储水器位置，直到在微量移液器尖端注意到微妙的正压。</li>
	<li>在相机操作软件中开始采集。打开 荧光快门。</li>
	<li>通过在控制面板中输入计算出的电压大小来吸出红细胞以达到所需的压力。 注意：抽吸红细胞的压力通常在 Δp = -5 至 -40 mmHg 的范围内。微量移液器吸头内应有明显的舌伸长（图2D）。</li>
	<li>在预设的时间内保持压力;然后，释放压力。</li>
	<li>移动微量移液器拾取下一个细胞并重复实验。</li>
</ol>7. 荧光强度分析
<ol><li>将保存的荧光图像加载到分析软件中。</li>
	<li>使用显示调整选项卡调整强度阈值。为此，可以手动输入值，也可以使用滑块，以确保荧光图像在分析软件中显示细胞的清晰对比度（参见补充文件1-补充图S1）。</li>
	<li>滚动到软件底部的时间线。找到指定的抽吸事件。</li>
	<li>单击 添加新曲面。定义分析 ROI。 注意：该软件提供了一个指导性的五步过程来调整和完成分割（参见 补充文件 1 - 补充图 S2 和 补充图 S3）。 注意：保持尽可能小的 ROI 以节省计算资源。</li>
	<li>使用背景减法滑块并使用滑块调整分割阈值，以获得最佳分割结果。 注意：这意味着除了抽吸事件外，应尽可能准确地分割背景（参见 补充文件 1 - 补充图 S4 和 补充图 S5）。</li>
	<li>添加 区域 滤波器以排除背景噪声（请参阅 补充文件 1 - 补充图 S6）。 注意：这是在后期处理阶段完成的。</li>
	<li>选择 统计 信息选项卡 | “详细 ”选项卡 | “平均值 ”选项卡。滚动以查找并选择强度平均值（请参阅 补充文件 1-补充图 S7）。</li>
	<li>将随时间变化的荧光信号迹线导出到.csv文件。</li>
	<li>打开导出的 csv 文件。从所有测量值中减去背景信号 Fb。</li>
	<li>使用公式（1）计算钙强度变化，ΔFmax： <img alt="Equation 1" src="/files/ftp_upload/66265/66265eq01.jpg" style="margin: auto;">（一） 其中 ΔFmax 是最大钙强度变化， Fb 是背景强度， F0 是静息强度。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66265/66265fig02.jpg"> 图 2：荧光偶联微量移液器抽吸组件。 （A） fMPA硬件系统概述，该系统将倒置显微镜与明场和荧光相机相结合。图像的左侧描绘了自制的水压计和控制箱，可以精确调节气动压力泵的压力。（B） 显微镜载物台，描绘了实验室和带有单个微量移液器的显微操纵器系统。（C） fMPA系统设置示意图。利用两个二向色镜对明场（黄色）和荧光（蓝色发射，绿色激发）信号进行同步成像，将光路从荧光光源（蓝色）引导到目标，然后引导到相机进行成像（绿色）。（D） 顶行表示明场图像，底行表示荧光图像。左图表示红细胞静止时微量移液器在抽吸前的位置。中间一栏记录了红细胞承受 -40 mmHg 负压的抽吸过程。右图描绘了经历负吸气压后的细胞形态。比例尺 = 5 μm。缩写：fMPA = 荧光耦合微量移液器抽吸;DM = 二向色镜;红细胞=红细胞。 <a href="https://www.jove.com/files/ftp_upload/66265/66265fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

使用荧光微量移液器吸液进行单细胞机械生物学研究

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J., Kuhr, F., Byfield, F., Levitan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette+aspiration+of+substrate-attached+cells+to+estimate+cell+stiffness.">Micropipette aspiration of substrate-attached cells to estimate cell stiffness.</a> Journal of Visualized Experiments. (67), e3886 (2012).</li><li>Rand, R. P., Burton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+red+cell+membrane.">Mechanical properties of the red cell membrane.</a> Biophysical journal. 4 (4), 303-316 (1964).</li><li>Hogan, B., Babataheri, A., Hwang, Y., Barakat, A. I., Husson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+cell+adhesion+by+using+micropipette+aspiration.">Characterizing cell adhesion by using micropipette aspiration.</a> Biophysical Journal. 109 (2), 209-219 (2015).</li><li>Henriksen, J. R., Ipsen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+membrane+elasticity+by+micro-pipette+aspiration.">Measurement of membrane elasticity by micro-pipette aspiration.</a> The European Physical Journal E. 14 (2), 149-167 (2004).</li><li>Hochmuth, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropipette+aspiration+of+living+cells.">Micropipette aspiration of living cells.</a> Journal of Biomechanics. 33 (1), 15-22 (2000).</li><li>Pu, H., 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=Micropipette+aspiration+of+single+cells+for+both+mechanical+and+electrical+characterization.">Micropipette aspiration of single cells for both mechanical and electrical characterization.</a> IEEE Transactions on Biomedical Engineering. 66 (11), 3185-3191 (2019).</li><li>Guevorkian, K., Ma&#238;tre, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+10+-+Micropipette+aspiration:+A+unique+tool+for+exploring+cell+and+tissue+mechanics+in+vivo.">Chapter 10 - Micropipette aspiration: A unique tool for exploring cell and tissue mechanics in vivo.</a> Methods in Cell Biology. , 187-201 (2017).</li><li>Biro, M., Ma&#238;tre, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14+-+Dual+pipette+aspiration:+A+unique+tool+for+studying+intercellular+adhesion.">Chapter 14 - Dual pipette aspiration: A unique tool for studying intercellular adhesion.</a> Methods in Cell Biology. 125, 255-267 (2015).</li></ol>

作者声明，他们没有关于本研究的竞争利益报告。

微量移液器抽吸法体现了一种精细的方法，部署了大量的压力调节、精确的空间协调和可靠的时间辨别，以探索细胞生物力学的深刻复杂性。本研究特别强调 fMPA 作为揭示红细胞在不同刺激下表现出的细微机械敏感反应的关键工具的应用。同时使用明场和荧光信号可以对细胞现象进行多方面的探索，从而推进细胞内钙流入的实时监测和检测。这种方法为红细胞的复杂机械传感反应提供了综合见解?...

细胞行为领域的不断发现强调了机械刺激（如张力、流体剪切应力、压缩和基质刚度）在决定动态细胞活动（如粘附、迁移和分化）中的作用。这些机械生物学方面对于阐明细胞如何与其生理环境相互作用并对其做出反应至关重要，从而影响各种生物过程 1,2。
在过去十年中，基于微量移液器的抽吸检测已成为研究各种细胞对机械刺激反应的多功能工具。该技术为单细胞水平上活细胞的内在机械特性提供了有价值的见解，包括细胞弹性模量、刚度和皮质张力。这些测定可以测量各种机械参数，例如细胞膜张力、施加在细胞膜上的压力和皮质张力（总结在表 1 中）。研究吸力丰富了我们对它们如何影响细胞功能和过程的理解，特别是在膜动力学领域，包括碎裂、伸长和萌芽 3,4。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>机械参数</td>
			<td>描述</td>
			<td>开创性方法</td>
		</tr><tr><td>电池刚度</td>
			<td>测量电池的机械刚度和弹性。</td>
			<td>细胞膜的抽吸和对负压的变形响应分析20,21。</td>
		</tr><tr><td>粘接强度</td>
			<td>评估细胞粘附在表面的强度。</td>
			<td>应用受控抽吸将粘附的细胞从底物上分离2,22。</td>
		</tr><tr><td>膜张力</td>
			<td>评估细胞膜内的张力或应力。</td>
			<td>膜变形响应的测量23,24.</td>
		</tr><tr><td>粘弹性</td>
			<td>表征细胞的粘性和弹性组合行为。</td>
			<td>抽吸时变形响应分析23,25.</td>
		</tr><tr><td>变形</td>
			<td>确定细胞改变形状的难易程度。</td>
			<td>受控吸力下变形程度的评估20,24.</td>
		</tr><tr><td>表面张力</td>
			<td>测量电池表面的张力。</td>
			<td>评估形成微量移液器膜突起所需的压力26.</td>
		</tr><tr><td>细胞-材料相互作用</td>
			<td>研究细胞与材料或底物之间的相互作用。</td>
			<td>与不同材料接触的细胞抽吸和相互作用的观察2,24。</td>
		</tr><tr><td>细胞间相互作用</td>
			<td>检查相邻细胞之间的相互作用。</td>
			<td>一组细胞的抽吸及其细胞间力的分析27.</td>
		</tr></tbody></table>表1：微量移液器抽吸法表征的机械参数。
基于微量移液器的抽吸技术已被广泛用于研究红细胞 （RBC），评估红细胞的变形性和各种机械特性，这对于了解它们在循环系统中的功能至关重要。红细胞表现出非凡的适应性，在穿过复杂的毛细血管网络和内皮间裂隙时保持其机械多功能性，防止变形5,6。在这个过程中，红细胞必须穿过狭窄至0.5-1.0μm的通道，承受多种机械力，包括拉伸和压缩7,8,9。它们对循环过程中血流产生的剪切应力也具有高度敏感性 10。这些过程促进了涉及钙内流的调节机制的激活，钙内流是一种关键的信号转导事件，在细胞对机械刺激的反应中具有公认的作用11,12。控制钙介导的机械感应的复杂机制仍然是正在进行的研究的引人注目的主题。
在这种情况下，fMPA是一种有效的方法，可以揭示在精确控制的机械力下钙的动员程度，允许同时应用机械调节（使用微量移液器抽吸系统）和可视化钙强度（使用荧光指示剂）。它特别模仿红细胞穿过狭窄血管时的生理场景。值得注意的是，我们开发的fMPA系统可以产生分辨率为1 mmHg的压力。实现的高速相机可以实现 100 毫秒的时间分辨率和亚微米级的空间分辨率。这些配置确保了对活细胞精确施加机械力，并同时捕获由此产生的细胞信号。此外，由于这种设置的集成工程性质，微量移液器抽吸法可以很容易地适应其他设备或技术，从而进一步探索细胞力学的复杂性。这种多功能性是这种方法的另一个优势。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#181;Manager</td>
			<td>Micro-Manager</td>
			<td></td>
			<td>Version 2.0.0</td>
		</tr>
		<tr>
			<td>1 mL Syringe&#160;</td>
			<td>Terumo</td>
			<td>210320D</td>
			<td>Cooperate with the Microfil&#160;</td>
		</tr>
		<tr>
			<td>200 &#181;L Pipette&#160;</td>
			<td>Eppendorf&#160;</td>
			<td>3123000055</td>
			<td>Red clood cell preparation</td>
		</tr>
		<tr>
			<td>22 x 40 mm Cover Slips</td>
			<td>Knittel Glass&#160;</td>
			<td>MS0014</td>
			<td>Cell chamber assembly</td>
		</tr>
		<tr>
			<td>50 mL Syringe&#160;</td>
			<td>Terumo</td>
			<td>220617E</td>
			<td>Connect to the water tower</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>Tryode&#39;s&#160; buffer preparation - 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2</td>
		</tr>
		<tr>
			<td>Centrifuge 5425</td>
			<td>Eppendorf&#160;</td>
			<td>5405000280</td>
			<td>Red clood cell preparation</td>
		</tr>
		<tr>
			<td>Clexane</td>
			<td>Sigma-Aldrich</td>
			<td>1235820</td>
			<td>To prevent clotting of the collected blood. 10,000 U/mL</td>
		</tr>
		<tr>
			<td>DAQami</td>
			<td>Diligent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence light source</td>
			<td>CoolLED</td>
			<td>pE-300</td>
			<td>Micropipette aspiration hardware system</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Narishige</td>
			<td>G-1</td>
			<td>Micropipette manufacture</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Tryode&#39;s&#160; buffer preparation - 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td>Tryode&#39;s&#160; buffer preparation - 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2</td>
		</tr>
		<tr>
			<td>High speed GigE camera</td>
			<td>Manta</td>
			<td>G-040B</td>
			<td>Micropipette aspiration hardware system</td>
		</tr>
		<tr>
			<td>High speed pressure clamp</td>
			<td>Scientific Instrument</td>
			<td>HSPC-2-SB</td>
			<td>Cooperate with the pressure pump</td>
		</tr>
		<tr>
			<td>High speed pressure clamp head stage&#160;</td>
			<td>Scientific Instrument</td>
			<td>HSPC-2-SB</td>
			<td>Cooperate with the pressure pump</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscopy&#160;</td>
			<td>Olympus&#160;</td>
			<td>Olympus IX83</td>
			<td>Micropipette aspiration hardware system</td>
		</tr>
		<tr>
			<td>Microfil&#160;</td>
			<td>World Precision Instruments&#160;</td>
			<td>MF34G-5</td>
			<td>34 G (67 mm Long) 
			Revome air bubble in the cut micropipette and test the opening of the pipette tip&#160;</td>
		</tr>
		<tr>
			<td>Micropipette Puller&#160;</td>
			<td>Sutter instrument</td>
			<td>P1000</td>
			<td>Micropipette manufacture&#160;</td>
		</tr>
		<tr>
			<td>Milli Q EQ 7000 Ultrapure Water Purification System</td>
			<td>Merck Millipore</td>
			<td>ZEQ7000T0C</td>
			<td>Carbonate/bicarbonate buffer &#38; Tryode&#39;s buffer preparation</td>
		</tr>
		<tr>
			<td>Pipette microforge&#160;</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Micropipette manufacture</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>Tryode&#39;s&#160; buffer preparation - 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2</td>
		</tr>
		<tr>
			<td>Pressue Pump&#160;</td>
			<td>Scientific Instrument</td>
			<td>PV-PUMP</td>
			<td>Induce controlled pressure during experiment</td>
		</tr>
		<tr>
			<td>Prime 95B Camera&#160;</td>
			<td>Photometrics</td>
			<td>Prime 95B sCMOS</td>
			<td>Flourscent imaging</td>
		</tr>
		<tr>
			<td>Rotary wheel remote unit&#160;</td>
			<td>Sensapex&#160;</td>
			<td>uM-RM3</td>
			<td>Control panel for micropipette position adjustment&#160;</td>
		</tr>
		<tr>
			<td>Scepter 3.0 Handheld Cell Counter</td>
			<td>Merck Millipore</td>
			<td>PHCC340KIT</td>
			<td>Automatic cell counter</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation - 2.65 g of NaHCO3 with 2.1 g of Na2CO3 in 250 mL of Mili Q water - Final pH = 8-9.</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td>Carbonate/bicarbonate buffer preparation - 2.65 g of NaHCO3 with 2.1 g of Na2CO3 in 250 mL of Mili Q water - Final pH = 8-9.</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Tryode&#39;s&#160; buffer preparation - 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic 
			Monohydrate (NaH2PO4 &#8226; H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>Tryode&#39;s&#160; buffer preparation - 12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, and 5.5 mM D-glucose supplemented with 1 mM CaCl2. Final pH = 7.2</td>
		</tr>
		<tr>
			<td>Touch screen control unit&#160;</td>
			<td>Sensapex&#160;</td>
			<td>uM-TSC</td>
			<td>Control panel for micropipette position adjustment&#160;</td>
		</tr>
		<tr>
			<td>X dry Objective&#160;</td>
			<td>Olympus&#160;</td>
			<td>Olympus 60x/0.70 LUCPlanFL</td>
			<td>Micropipette aspiration hardware system</td>
		</tr>
	</tbody>
</table>

fluorescence micropipette aspiration assay to investigate red blood cell mechanosensing

长期以来，微量移液器抽吸法一直是研究活细胞力学的基石，为细胞对机械应力的反应提供了见解。本文详细介绍了荧光偶联微量移液器抽吸 （fMPA） 测定的创新应用。fMPA 检测引入了管理精确机械力的能力，同时监测由离子通道介导的活细胞机械转导过程。精密的装置包括一个精密设计的硼硅酸盐玻璃微量移液器，连接到一个精细调节的储水器和气动吸气系统，便于以± 1 mmHg的增量进行受控压力的应用。一个显著的改进是落射荧光成像的集成，允许在抽吸过程中同时观察和定量细胞形态变化和细胞内钙通量。fMPA检测通过其落射荧光成像与微量移液器抽吸的协同组合，为在具有挑战性的机械环境中研究细胞机械传感设定了新标准。这种多方面的方法适用于各种实验设置，为单细胞机械传感机制提供了重要的见解。

为了建立微量移液器抽吸测定，我们首先构建了一个定制的细胞室，该室由两个金属方块（铜/铝）组成，通过手柄连接。粘贴两个第三切割的玻璃盖玻片（40 mm × 7 mm × 0.17 mm），以创建一个充满悬浮在 Tyrode 缓冲液中的 200 μL RBC 的腔室。将红细胞引入腔室后，将定制的硼硅酸盐微量移液器固定在支架上，并使用微量操纵器小心地放置在腔室内。随后，将微量移液器拉近以捕获目标红细胞。
<p c...

Watch this Scientific Journal Video about Advancing Live-Cell Mechanobiology Through Fluorescence Microaspiration at JoVE.com

作者聚焦：通过荧光显微抽吸推进活细胞力学生物学

探索机械应力下的细胞行为对于细胞力学和机械生物学的进步至关重要。我们介绍了荧光微量移液器抽吸（fMPA）技术，这是一种将受控机械刺激与单细胞细胞内信号传导综合分析相结合的新方法。该技术研究了活细胞机械生物学的新深入研究。

荧光微量移液器抽吸法研究红细胞机械传感

Micropipette aspiration assays have long been a cornerstone for the investigation of live-cell mechanics, offering insights into cellular responses to ...

我们的研究范围是针对生活方式机械生物学，特别是施加机械应力的细胞的行为。我们正在研究单细胞中的细胞内信号传导。所提出的FMPA系统利用荧光成像与机械刺激相结合。即吸气压力。目前有进一步的进展，试图将两种以上的技术与微吸装置（如微流体或图像分析软件）相结合。目前的实验挑战在于，该设置非常劳动密集型，并且依赖于操作员。由于系统是手动操作的，因此在特定步骤中会出现不一致的情况。例如，灯丝预热。研究结果显示，当压力在负10毫米汞柱至负40毫米汞柱之间逐渐增加时，钙离子流入红细胞的量也相应增加。这表明红细胞具有感知其机械环境变化的能力，并通过快速的钙相关通道活动做出反应。本研究特别强调 FMPA 作为揭示红细胞在不同刺激下表现出的细微机械敏感反应的关键工具的应用。

fluorescence-micropipette-aspiration-assay-to-investigate-red-blood

Research

JoVE Journal

Biology

Fluorescence Micropipette Aspiration Assay to Investigate Red Blood Cell Mechanosensing

717 次观看.  The University of Sydney. 我们的研究范围是针对生活方式机械生物学，特别是施加机械应力的细胞的行为。我们正在研究单细胞中的细胞内信号传导。所提出的FMPA系统利用荧光成像与机械刺激相结合。即吸气压力。目前有进一步的进展，试图将两种以上的技术与微吸装置（如微流体或图像分析软件）相结合。目前的实验挑战在于，该设置非常劳动密集型，并且依赖于操作员。由于系统是?...

视频: 作者聚焦：通过荧光显微抽吸推进活细胞力学生物学

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

斑马鱼的响应行为的检测，以测量光强的变化

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

科研

生物学

Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) procedure. In the past, we standardized a model consisting of latex glove containing fresh cattle liver for practicing FNAB. However, this model is difficult to organize and prepare on short notice, with the procurement of fresh cattle liver being the most challenging aspect. Handling of liver with contamination-related problems is also a significant draw back. In addition, the glove material leaks after a few needle passes, with resulting mess.
We have established a novel simple method of embedding a small piece of sausage or banana in a commercially available silicone rubber caulk. This model allows the retention of vacuum seal and aspiration of material from the embedded specimen, resembling an actual FNAB procedure on clinical mass lesions.
The aspirated material in the needle hub can be processed similar to the specimens procured during an actual FNAB procedure, facilitating additional proficiency in smear preparation and staining.

Practicing of fine needle aspiration biopsies (FNAB) by trainees is relatively challenging, due to the lack of an easily available, appropriate lesion. Preparation of an AV phantom lesion for practicing the FNAB procedure and mastering proficiency is relatively easy.

制备及使用幻影病变实践细针穿刺活检

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

为评估细胞Mechanosensing和Durotaxis简化系统<em&gt;体外</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

活体细胞荧光显微镜观察微生物细胞生长过程中的基本过程

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

用螺旋粘度计对人血小板和细胞表面受体进行均匀剪切检测

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...