这项工作部分得到加拿大自然科学和工程研究理事会（NSERC）和加拿大卫生研究院（CIHR）的资助。感谢温尼伯维多利亚维多利亚综合医院和温尼伯七橡综合医院临床应用研究与教育研究所，负责管理人类临床样本。我们感谢Hagit Peretz-Soroka博士有关测定操作策略的有益讨论。 <style type="text/css">
</style>我们非常感谢滑铁卢大学的卡罗琳教授和陈晓明博士在拍摄过程中的慷慨支持。

所有人类样品采集方案均由温尼伯曼尼托巴大学联合学院研究伦理委员会批准。 微流体装置制造（ 图1A ） <ol><li> 设计和打印透明面具。 <ol><li>设计设备如前所述25 。参见图1A 。 注意:设备包括两层。第一层（4μm高）限定了细胞对接屏障通道，以将细胞捕获在梯度通道旁边。第二层（60μm高）定义了梯度产生通道，电池负载的端口和通道，化学品入口容器和废物出口。对准标记设计用于两层。对于第二层，上游蛇形输入通道的长度和宽度分别为60mm和200μm;下游的长度和宽度蛇形输入通道分别为6 mm和280μm。 </li><li>使用高分辨率打印机将第一和第二层功能打印到透明蒙版。 注意:打印分辨率取决于设计中的最小功能。在目前的设计中，选择了32,000 dpi，最小为10μm。 </li></ol></li><li> 清洁硅片。 <ol><li>将硅片放入等离子体清洁机中。施加真空3分钟。 </li><li>打开等离子电源并将电平设置为高电平。使用氧等离子体处理硅晶片30分钟。 </li><li>关闭等离子体清洁器并取出硅片;硅晶片准备用于制造器件模具。 </li></ol></li><li> 在洁净室中通过光刻制造第一层。 注意:精确的制造参数可能因加工设备而异。 <ol><li>稀释10 mL SU-8 2025，用10 mL SU-8 2000在玻璃破碎机中制备设计的光致抗蚀剂。将混合物放在通风橱中10分钟，直到气泡消失。 </li><li>用合适的卡盘小心地将清洁的硅晶片放在旋转器上，并施加真空以固定硅晶片。 </li><li>小心地将3mL的光致抗蚀剂混合物倒入硅晶片的中心。以500 rpm旋转5 s。然后以3,000rpm旋转30秒以获得硅晶片上的最终4μm厚度的光致抗蚀剂涂层。 </li><li>小心地从旋转器中取出硅晶片，并在95℃下将硅晶片在电热板上烘烤4分钟。 </li><li>将硅晶片小心地放置在掩模对准器上，并将UV曝光时间设置为6 s。使用胶带小心地将第一层的透明面具贴在透明玻璃板上。 </li><li>将带有附着面罩的透明玻璃板轻轻放置在对准器上，并将面罩与硅晶片对准。暴露silicon晶圆到UV以图案化细胞对接结构。 </li><li>小心地取出玻璃板并取出暴露的硅晶片。在95℃将硅片烘烤4分钟。 </li><li>将硅晶片转移到通风橱，并将其放入含有SU-8显影剂的玻璃盘中。轻轻摇动30秒。 </li><li>使用新鲜SU-8显影剂，然后通风柜内的异丙醇（IPA）清洁硅晶片。通风柜内的氮气干燥硅晶片;第一层准备好了。 </li></ol></li><li> 在第一层制作第二层。 <ol><li>使用胶带覆盖第一层上的对准标记。将具有第一层的硅晶片小心地放置在旋转器的真空吸盘上，并施加真空以固定硅晶片。 </li><li>在硅晶片上倒入3 mL的SU-8 2025光致抗蚀剂。以500rpm将硅晶片旋转5秒。然后以2000rpm旋转30分钟以在硅晶片上获得最终的60μm厚度的光致抗蚀剂涂层。 </li><li>小心地将硅晶片从旋转器中取出并将其转移到电热板;在65℃烘烤2分钟。 </li><li>轻轻取出胶带以露出第一层上的对准标记。将硅片放在电热板上，在95℃烘烤6分钟。 </li><li>小心地将硅晶片放置在掩模对准器上，并将UV曝光时间设置为18秒。 </li><li>使用胶带小心地将第二层的透明掩模贴在透明玻璃板上。 </li><li>小心地将附有面罩的玻璃板放在校准器上，并使用对准器的检查显微镜，通过交叉对准标记将掩模和硅晶片上的第一层对准。 </li><li>将光致抗蚀剂涂覆的硅晶片暴露于UV以对电池负载和梯度通道进行图案化。 </li><li>小心取出玻璃板，取出硅胶AFER。将硅晶片在65℃的热板上烘烤2分钟，然后将硅晶片转移到另一个95℃的热板上并烘烤6分钟。 </li><li>将硅晶片转移到通风橱，并将其放入含有SU-8显影剂的玻璃盘中。轻轻摇动6分钟。 </li><li>使用新鲜的SU-8显影剂，然后在通风橱内通过IPA清洁硅晶片。 </li><li>使用通风柜内的氮气干燥硅晶片。将硅晶片置于电热板上，并在150℃下硬模烘30分钟;第二层就绪。 </li></ol></li><li> 主模表面改性。 注意:将硅烷化表面改性步骤应用于SU-8模具以便于在软光刻中从模具中脱离聚二甲基硅氧烷（PDMS）。 <ol><li>将10μL十三氟-1,1,2,2-四羟基辛基（三氯硅烷）溶液置于微量移液管尖端。将微量移液器吸头放入15 mL塑料管中请松开管子的盖子。 </li><li>将管和SU-8图案化的硅晶片放置在干燥器内并施加真空1小时;面罩模具准备好用于制造PDMS设备。 </li></ol></li><li> 制造PDMS设备。 <ol><li>通过在塑料烧杯中混合40g PDMS碱和4g固化剂来制备PDMS溶液。将准备好的SU-8母模置于培养皿中，小心地将44g PDMS溶液倒入模具中。 </li><li>将培养皿放置在干燥器中，并施加真空以使PDMS溶液脱气20分钟。然后将培养皿放入烤箱中，并在80℃下将PDMS固化2小时。 </li><li>烘烤后，取出培养皿，放在干净的工作台上。从SU-8模具小心切割和剥离PDMS板。 </li><li>使用3mm直径的冲孔机冲出电池加载端口。使用6毫米直径的冲孔机冲出化学品入口容器和废物出口。 </li><li>清除上的灰尘使用胶带的PDMS板的表面。将PDMS板和干净的玻璃片放入等离子机中。应用真空3分钟。 </li><li>打开等离子电源并将电平设置为高电平。轻轻调整空气阀和等离子体处理PDMS板和玻璃板3分钟。 </li><li>关闭等离子体电源并释放真空。用镊子小心取出PDMS板和玻片。 </li><li>立即将PDMS板（通道结构面朝下）放在玻片的顶部;轻轻按下PDMS板将其固定在玻璃上。立即用去离子水填充微流体通道;完成微流体装置的制造和组装。 </li></ol></li></ol>微流控细胞迁移测定法<ol><li> 微流体装置制备。 <ol><li>通过将50μL的股票纤连蛋白溶液（1mg / mL）稀释至950来制备50μg/ mL纤连蛋白溶液81; L Dulbecco的磷酸盐缓冲盐水（DPBS）在生物安全柜内。 </li><li>通过将9mL Roswell Park Memorial Institute培养基（RPMI-1640）和1mL含4％牛血清白蛋白（BSA）的RPMI-1640混合来制备迁移培养基。 </li><li>从设备中取出去离子水。 </li><li>从出口向设备中加入100μL纤连蛋白溶液。等待3分钟，以确保所有通道都充满纤连蛋白溶液。将微流体装置放置在覆盖的培养皿中，室温下1小时。 </li><li>从设备中取出纤连蛋白溶液。从出口添加100μL迁移介质。等待3分钟以确保所有通道都填充有迁移介质。 </li><li>在室温下再孵育1小时;然后该装置准备进行趋化实验。 </li></ol></li><li> 化学吸附剂溶液制备趋化实验。 <ol><li>在t中制备100nM fMLP溶液口服1mL迁移培养基。将5μL含有FITC-葡聚糖（10kDa，1mM）的溶液与1.5mL管中的fMLP溶液混合。 注意:FITC-Dextran用于梯度测量。或者，使用罗丹明作为梯度指示剂。然后将fMLP化学引诱剂溶液准备用于趋化实验。 </li></ol></li><li> 痰样品制备。 注意:通过COPD患者的痰样品梯度诱导的嗜中性粒细胞趋化性作为这种全片式方法的临床诊断应用进行了测试。 <ol><li>获得人类道德规范来收集COPD患者的痰样品。 注意:我们获得批准在温尼伯的七橡综合医院收集样品（由马尼托巴大学批准）。 </li><li>从所有科目获取知情的书面同意书。 </li><li>收集COPD患者的自发性痰样品。将500μL痰样品放入1.5 mL管中。 </li><li>加入500μL0.1％di硫代苏糖醇在1.5 mL管中轻轻混匀。将管置于37℃的水浴中15分钟。 </li><li>将样品以753 xg离心10分钟，然后收集上清液。以865xg离心上清5分钟，然后收集最终上清液。将收集的上清液储存在-80°C冷冻箱中，然后再使用。 </li><li>准备进行趋化性实验时，解冻痰液;将900μL迁移培养基转移到1.5 mL管中，并与生物安全柜内的100μL痰液混合;然后痰液可用于趋化实验。 </li></ol></li><li> 血样收集。 <ol><li>获得人类道德规范，从健康捐赠者收集血液样本。从所有献血者处获得知情的书面同意书。 注意:这些样品是在温尼伯维多利亚综合医院获得的（由马尼特大学联合研究伦理委员会批准OBA）。 </li><li>通过静脉穿刺收集血液样品，并将样品放入EDTA涂层的管中。在实验之前将管保持在生物安全柜中。 </li></ol></li></ol> 3.片上趋势测定操作<ol><li> 片上细胞分离（ 图1B ）。 <ol><li>将10μL全血置于生物安全柜内的1.5 mL管内。 注意:血液采集的细节在2.4节。 </li><li>从嗜中性粒细胞分离试剂盒（参见材料表）中加入2μL抗体混合物（Ab）和2μL磁性颗粒（MP），放入1.5 mL管中，轻轻混匀;这将标记血液中的细胞，除了嗜中性粒细胞。 </li><li>在室温下孵育血液中的Ab-MP混合物5分钟。 注意:这将磁性标记血液中标记抗体的细胞。 </li><li>将两个小磁盘连接到电池单元的两侧ort的设备。从设备的所有端口吸出介质。 </li><li>慢慢地从细胞装载端口将2μL血液 - Ab-MP混合物移液到微流体装置中。 注意:磁性标记的细胞被捕获到细胞加载端口的侧壁，而嗜中性粒细胞将流入该装置并被捕获在细胞对接结构上。 </li><li>等待几分钟，直到足够的嗜中性粒细胞被困在细胞对接区域。 </li></ol></li><li> 趋化性测定（ 图1C ）。 <ol><li>将微流控装置置于37℃的温度控制显微镜下。 </li><li>使用两个移液器将100μL化学引诱物溶液（fMLP或痰液）和100μL迁移培养基添加到其指定的入口储存器中;这将通过由加压辅助的连续层流化学混合在梯度通道中产生化学引诱剂梯度e平衡结构。 注意:COPD患者的痰液收集细节在2.3节。 <ol><li>对于中等对照实验，仅向两个入口储层添加迁移介质。 </li></ol></li><li>获得梯度通道中FITC-葡聚糖的荧光图像。 </li><li>使用命令"File | Open"将图像导入ImageJ软件。 </li><li>使用命令"Analyze | Plot Profile"测量梯度通道上的荧光强度曲线。 </li><li>将测量数据导出到电子表格以进一步绘制。 </li><li>在温度控制的显微镜阶段或常规细胞培养箱中孵育该装置15分钟。 </li><li>使用10X目标对梯度通道进行成像，以记录单元格的最终位置进行数据分析。 </li><li>如果需要，通过延时显微镜记录设备中的细胞迁移。 </li></ol></li></ol>细胞迁移Data分析（ 图1C ） <ol><li>通过计算从对接结构的细胞迁移距离来分析趋化性测定，如下所述。参见图1C 。 </li><li>将图像导入NIH ImageJ软件（版本1.45）。 </li><li>选择移动到渐变通道的每个单元格的中心。 </li><li>测量所选单元格的最终位置的坐标。测量对接结构边缘处的点的坐标作为初始参考位置。 </li><li>将测量的坐标数据导出到电子表格软件（ 例如 Excel）。计算细胞的迁移距离，作为细胞的最终位置与沿着梯度方向的初始参考位置之间的差异。 </li><li>校准到千分尺的距离。计算所有细胞的迁移距离的平均值和偏差作为趋化性的量度。 </li><li>比较迁移在使用Student&#39;s t检验的介质对照实验中存在化学引诱剂梯度时的配给距离。 </li><li>如果记录细胞迁移的延时图像，可以通过细胞跟踪分析15进一步分析细胞迁移和趋化性。 注意:构建和执行片上趋化性测定所需的材料详见材料表。 </li></ol>

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Y., Aranyosi, A. J., Toner, M., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+decisions+during+neutrophil+chemotaxis+inside+bifurcating+channels.">Directional decisions during neutrophil chemotaxis inside bifurcating channels.</a> Integr Biol. 2 (11-12), 639-647 (2010).</li><li>Jones, C. N., 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=Spontaneous+neutrophil+migration+patterns+during+sepsis+after+major+burns.">Spontaneous neutrophil migration patterns during sepsis after major burns.</a> PloS One. 9 (12), e114509 (2014).</li><li>Butler, 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=Burn+injury+reduces+neutrophil+directional+migration+speed+in+microfluidic+devices.">Burn injury reduces neutrophil directional migration speed in microfluidic devices.</a> PloS One. 5 (7), e11921 (2010).</li><li>Wu, 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=A+microfluidic+platform+for+evaluating+neutrophil+chemotaxis+induced+by+sputum+from+COPD+patients.">A microfluidic platform for evaluating neutrophil chemotaxis induced by sputum from COPD patients.</a> PloS One. 10 (5), e0126523 (2015).</li><li>Sackmann, E. 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=Microfluidic+kit-on-a-lid:+a+versatile+platform+for+neutrophil+chemotaxis+assays.">Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays.</a> Blood. 120 (14), e45-e53 (2012).</li><li>Agrawal, N., Toner, M., Irimia, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+migration+assay+from+a+drop+of+blood.">Neutrophil migration assay from a drop of blood.</a> Lab Chip. 8 (12), 2054-2061 (2008).</li><li>Jones, C. N., 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=Microfluidic+platform+for+measuring+neutrophil+chemotaxis+from+unprocessed+human+whole+blood.">Microfluidic platform for measuring neutrophil chemotaxis from unprocessed human whole blood.</a> J Vis Exp. (88), (2014).</li><li>Jones, C. N., 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=Microfluidic+assay+for+precise+measurements+of+mouse,+rat,+and+human+neutrophil+chemotaxis+in+whole-blood+droplets.">Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets.</a> J Leukocyte Biol. 100 (1), 241-247 (2016).</li><li>Sackmann, E. K. -. 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=Characterizing+asthma+from+a+drop+of+blood+using+neutrophil+chemotaxis.">Characterizing asthma from a drop of blood using neutrophil chemotaxis.</a> P Natl Acad Sci. 111 (16), 5813-5818 (2014).</li><li>Wu, 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=An+all-on-chip+method+for+testing+neutrophil+chemotaxis+induced+by+fMLP+and+COPD+patient's+sputum.">An all-on-chip method for testing neutrophil chemotaxis induced by fMLP and COPD patient's sputum.</a> Technology. 04 (02), 104-109 (2016).</li></ol>

没有利益冲突的披露。

在本文中，描述了在单个微流体芯片上直接从全血中分离嗜中性粒细胞并进行趋化性试验的详细方案。该方法易于操作，阴性选择高纯度中性粒细胞，快速样品至结果趋化性测试，减少试剂和样品消耗，以及精确的细胞迁移数据分析，提供了有用的功能。粗略估计，来自输入全血样本的嗜中性粒细胞的至少25％有效地进入装置中的对接结构，并且通过片上吉姆萨染色发现嗜中性粒细胞纯度高。 ...

趋化因子是一种有针对性的细胞迁移到可溶性化学物质浓度梯度的过程，在很多生物学过程中都是非常重要的，包括免疫反应1,2,3 ，组织发育4和癌症转移5 。嗜中性粒细胞是最丰富的白细胞亚群，在使身体的先天宿主防御功能以及介导适应性免疫反应6,7中发挥关键作用。嗜中性粒细胞配备有高度调节的趋化机制，允许这些运动免疫细胞通过化学疗法8对病原体衍生的化学引诱物（ 例如 fMLP）和宿主衍生的化学引诱物（ 例如白细胞介素-8）作出反应。嗜中性粒细胞迁移和趋化性介导各种生理问题和疾病如炎症和癌症1,9 。因此，准确评估嗜中性粒细胞趋化性为研究嗜中性粒细胞生物学和相关疾病提供了重要的功能读数。 与广泛使用的常规趋化性测定（ 例如 transwell测定10 ）相比，由于精确控制的化学梯度产生和小型化，微流控装置显示出对定量评估细胞迁移和趋化性的巨大前景11,12,13 。在过去二十年左右，已经开发出各种微流体装置来研究不同生物细胞类型，特别是中性粒细胞11的趋化性。大量的努力是用于描述时空复合物中嗜中性粒细胞迁移的特征<html>配置在微流体装置14,15中的液晶梯度。还开发了有趣的策略来研究使用微流体装置的嗜中性粒细胞的定向决策16。从生物学方面研究，微流体装置的应用已经扩展到临床样本用于疾病评估17,18,19 。然而，许多微流体装置的使用仅限于专门的研究实验室，并且需要从大量血液样品中获得长时间的嗜中性粒细胞分离。因此，直接从一滴全血20,21,22 ，ef"&gt; 23,24。 朝着这个方向，开发了一种全面的片上方法，其将磁性中性粒细胞纯化和随后的趋化性测定整合到单个微流体装置25上 。这种全片式方法具有以下新颖的特征:1）与以前的片上策略相比，通过基于粘附的细胞捕获或基于细胞大小的滤波来分离嗜中性粒细胞20,22 ，这种新方法允许高纯度，中性粒细胞与小体积全血的片上磁分离，以及化学诱导剂刺激时的趋化性测量; 2）细胞对接结构有助于使嗜中性粒细胞的初始位置靠近化学梯度通道，并允许简单的趋化性分析，而无需单细胞追踪; 3）中和粒细胞分离和chemot的整合在单个微流体装置上的轴测定允许在实验步骤之间没有中断的25分钟内快速的样品 - 结果趋化性分析。 本文提供了一个详细的协议，用于这种全片式趋化性测定的构建，操作和数据分析方法。该文件证明了通过测试已知的重组化学引诱物和来自患者的复杂趋化性样品来进行中性粒细胞趋化性的开发方法的有效使用，随后关于故障排除策略，限制和未来方向的讨论。

<table><tbody><tr><td>&lt;strong&gt;Device fabrication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mask aligner</td><td>ABM</td><td>N/A</td><td></td></tr><tr><td>Spinner</td><td>Solitec</td><td>5000</td><td></td></tr><tr><td>Hotplate</td><td>VWR</td><td>11301-022</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-001</td><td></td></tr><tr><td>Vacuum dessicator</td><td>Fisher Scientific</td><td>08-594-15A</td><td></td></tr><tr><td>Digital scale</td><td>Ohaus</td><td>CS200</td><td></td></tr><tr><td>SU-8 2000 thinner</td><td>Microchem</td><td>SU-8 2000</td><td></td></tr><tr><td>SU-8 2025 photoresist</td><td>Microchem</td><td>SU-8 2025</td><td></td></tr><tr><td>SU-8 developer</td><td>Microchem</td><td>SU-8 developer</td><td></td></tr><tr><td>Si wafer</td><td>Silicon, Inc</td><td>LG2065</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>Fisher Scientific</td><td>A416-4</td><td></td></tr><tr><td>(tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane</td><td>Gelest</td><td>78560-45-9</td><td></td></tr><tr><td>Polydimethylsiloxane&lt;br/&gt; (PDMS)</td><td>Ellsworth Adhesives</td><td>2065622</td><td></td></tr><tr><td>Petri Dish</td><td>Fisher Scientific</td><td>FB0875714</td><td></td></tr><tr><td>Glass slides</td><td>Fisher Scientific</td><td>12-544-4</td><td></td></tr><tr><td>Cutting pad</td><td>N/A</td><td>N/A</td><td>Custom-made</td></tr><tr><td>Punchers</td><td>N/A</td><td>N/A</td><td>Custom-made</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Source&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;On-chip cell isolation and chemotaxis assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640</td><td>Fisher Scientific</td><td>SH3025502</td><td></td></tr><tr><td>DPBS</td><td>Fisher Scientific</td><td>SH3002802</td><td></td></tr><tr><td>Bovine serum albumin&lt;br/&gt; (BSA)</td><td>Sigma-Aldrich</td><td>SH3057402</td><td></td></tr><tr><td>Fibronectin</td><td>VWR</td><td>CACB356008</td><td></td></tr><tr><td>fMLP</td><td>Sigma-Aldrich</td><td>F3506-10MG</td><td></td></tr><tr><td>Magnetic disks</td><td>Indigo Instruments</td><td>44202-1</td><td>5 mm in diameter,&lt;br/&gt; 1 mm thick</td></tr><tr><td>FITC-Dextran</td><td>Sigma-Aldrich</td><td>FD10S</td><td></td></tr><tr><td>Rhodamine</td><td>&lt;br/&gt; Sigma-Aldrich</td><td>R4127-5G</td><td></td></tr><tr><td>Giemsa stain solution</td><td>Rowley Biochemical Inc.</td><td>G-472-1-8OZ</td><td></td></tr><tr><td>EasySep Direct Human&lt;br/&gt; Neutrophil Isolation&lt;br/&gt; Kit</td><td>STEMCELL&lt;br/&gt; Technologies Inc</td><td>19666</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Nikon Ti-U inverted fluorescent microscope</td><td>Nikon</td><td>Ti-U</td><td></td></tr><tr><td>Microscope environmental chamber.</td><td>InVivo Scientific</td><td>N/A</td><td></td></tr><tr><td>CCD camera</td><td>Nikon</td><td>DS-Fi1</td><td></td></tr></tbody></table>

an all-on-chip method for rapid neutrophil chemotaxis analysis directly from a drop of blood

嗜中性粒细胞迁移和趋化性对我们身体的免疫系统至关重要。微流控装置越来越多地用于调查嗜中性粒细胞迁移和趋化性，因为它们具有实时可视化，精确控制化学浓度梯度产生以及降低的试剂和样品消耗的优点。最近，微流体研究人员正在越来越多地努力开发直接从全血中开发整合且易于操作的微流体趋化性分析系统。在这个方向上，第一个全芯片方法被开发用于整合嗜中性粒细胞的磁性负性纯化和来自小血容量样品的趋化性测定。这种新方法允许在25分钟内快速进行样品至结果嗜中性粒细胞趋化性测试。在本文中，我们提供了详细的构建，操作和数据分析方法，用于片上趋化性测定，讨论故障排除策略，limi重点和未来发展方向。显示了使用这种全芯片方法测试定义的化学引诱物， N-甲基-Mult-Leu-Phe（fMLP）和来自慢性阻塞性肺疾病（COPD）患者的痰的中性粒细胞趋化性测定的代表性结果。该方法适用于许多细胞迁移相关调查和临床应用。

嗜中性粒细胞直接在微流体装置中从一滴全血中选择。通过片上吉姆萨染色验证了分离的嗜中性粒细胞的纯度，结果显示中性粒细胞典型的环状和叶状核（ 图2A ） 25 。这表明从小体积的全血中可以获得高纯度的片上嗜中性粒细胞分离。此外，对接结构可以在施加化学梯度（ 图2B ） 25之前有?...

Watch this Scientific Journal Video about An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood at JoVE.com

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

本文提供了通过在单个微流体芯片上整合来自全血的片上嗜中性粒细胞分离和趋化性测试来进行快速嗜中性粒细胞趋化性测定的详细方法。

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

an-all-on-chip-method-for-rapid-neutrophil-chemotaxis-analysis

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

17.0K 次观看.  Chinese Academy of Sciences. 本文提供了通过在单个微流体芯片上整合来自全血的片上嗜中性粒细胞分离和趋化性测试来进行快速嗜中性粒细胞趋化性测定的详细方法。 

视频: An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

一个微流控芯片单细胞的多功能化学分析

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

科研

生物工程

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

免疫与感染

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

人中性粒细胞中 β2 整合素激活抑制剂的筛选

A Flow Cytometry-Based High-Throughput Technique for Screening Integrin-Inhibitory Drugs

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing conformational-change-reporting antibodies and high-throughput flow cytometry. The method can also serve as a guide for other antibody-based high-throughput screening methods. &#946;2 integrins are leukocyte-specific adhesion molecules that are crucial in immune responses. Neutrophils rely on integrin activation to exit the bloodstream, not only to fight infections but also to be involved in multiple inflammatory diseases. Controlling &#946;2 integrin activation presents a viable approach for treating neutrophil-associated inflammatory diseases. In this protocol, a monoclonal antibody, mAb24, which specifically binds to the high-affinity headpiece of &#946;2 integrins, is utilized to quantify &#946;2 integrin activation on isolated primary human neutrophils. N-formylmethionyl-leucyl-phenylalanine (fMLP) is used as a stimulus to activate neutrophil &#946;2 integrins. A high-throughput flow cytometer capable of automatically running 384-well plate samples was used in this study. The effects of 320 chemicals on &#946;2 integrin inhibition are assessed within 3 h. Molecules that directly target &#946;2 integrins or target molecules in the G protein-coupled receptor-initiated integrin inside-out activation signaling pathway can be identified through this approach.

作者聚焦:鉴定 β2 整合素激活小分子拮抗剂的方法的开发 

This protocol describes a flow cytometry-based, high-throughput screening method to identify small-molecule drugs that inhibit &#946;2 integrin activation on human neutrophils.

一种基于流式细胞术的高通量技术筛选整合素抑制药物

This protocol aims to establish a method for identifying small molecular antagonists of &#946;2 integrin activation, utilizing ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

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一个微流控芯片单细胞的多功能化学分析

微流体平台从未经加工的全血测定中性粒细胞趋

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人中性粒细胞流动室粘附实验

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

一种基于流式细胞术的高通量技术筛选整合素抑制药物

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60