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 border="0" cellpadding="0" cellspacing="0" width="581">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:581px;">Device fabrication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mask aligner</td>
			<td>ABM</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spinner</td>
			<td>Solitec</td>
			<td>5000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>VWR</td>
			<td>11301-022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuum dessicator</td>
			<td>Fisher Scientific</td>
			<td>08-594-15A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Digital scale</td>
			<td>Ohaus</td>
			<td>CS200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 2000 thinner</td>
			<td>Microchem</td>
			<td>SU-8 2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 2025 photoresist</td>
			<td>Microchem</td>
			<td>SU-8 2025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Microchem</td>
			<td>SU-8 developer</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Si wafer</td>
			<td>Silicon, Inc</td>
			<td>LG2065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">isopropyl alcohol</td>
			<td>Fisher Scientific</td>
			<td>A416-4</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:159px;">(tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane</td>
			<td>Gelest</td>
			<td>78560-45-9</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Polydimethylsiloxane 
			(PDMS)</td>
			<td>Ellsworth Adhesives</td>
			<td>2065622</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting pad</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Punchers</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">Name </td>
			<td>Source</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">On-chip cell isolation and chemotaxis assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">RPMI 1640</td>
			<td>Fisher Scientific</td>
			<td>SH3025502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>Fisher Scientific</td>
			<td>SH3002802</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Bovine serum albumin 
			(BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>SH3057402</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Fibronectin</td>
			<td>VWR</td>
			<td>CACB356008</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">fMLP</td>
			<td>Sigma-Aldrich</td>
			<td>F3506-10MG</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Magnetic disks</td>
			<td style="width:124px;">Indigo Instruments</td>
			<td>44202-1</td>
			<td style="width:164px;">5 mm in diameter, 
			1 mm thick</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:159px;">FITC-Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD10S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"> Rhodamine</td>
			<td> 
			Sigma-Aldrich</td>
			<td>R4127-5G</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Giemsa stain solution</td>
			<td style="width:124px;">Rowley Biochemical Inc.</td>
			<td style="width:135px;">G-472-1-8OZ</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:159px;">EasySep Direct Human 
			Neutrophil Isolation 
			Kit</td>
			<td style="width:124px;">STEMCELL 
			Technologies Inc</td>
			<td>19666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:159px;">Dithiothreitol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:159px;"> Nikon Ti-U inverted fluorescent microscope </td>
			<td style="width:124px;">Nikon</td>
			<td>Ti-U</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:159px;">Microscope environmental chamber.</td>
			<td style="width:124px;">InVivo Scientific</td>
			<td>N/A</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:159px;"> CCD camera</td>
			<td style="width:124px;">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

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 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

一个1.5小时的程序，鉴定肠球菌直接从血培养的物种

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

科研

免疫与感染

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

活体显微镜的脾:寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

为快速细菌鉴定和药敏试验的准备血培养微丸

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

对于血液处理快速的方法，以提高血浆肽水平的人血产量

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...