The authors would like to thank the following funding sources: RO1 DA 12104, RO1 DA 022935, RO1 DA031202, K05DA033881, P50 DA 011806, 1R01DA034582 (to S.R) and F31 DA026264-01A1, T32 DA07097 (to J.N.).

注意:此协议可以如在我们先前发表的作品12使用用于多种应用，如吞噬作用和肌动蛋白聚合的定量。的协议适合于各种变形例， 表1列出的细胞类型和颗粒在过去的研究中成功地使用。规范使用该协议的吞噬作用或肌动蛋白聚合的定量说明了图1。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/52195/52195diagram1.jpg" width="600" /> 图1:荧光分析对吞噬作用和肌动蛋白聚合的量化的图后，将细胞铺板，处理，并使其附着，标有绿色荧光素（FITC）的粒子加入到细胞吞噬作用。反应通过锥虫或antibi停止耳部清洗（以排除非内化的颗粒），并固定在4％多聚甲醛进行。细胞随后用红色荧光标记的肌动蛋白（罗丹明鬼笔）和蓝色荧光标记（DAPI）。吞噬作用和肌动蛋白聚合的索引被量化为绿/蓝（FITC / DAPI），或红/蓝（罗丹明/ DAPI）荧光的相对荧光单位的比率。 1.电镀和细胞治疗注:在开始之前，考虑与至少4技术重复运行的治疗，并包括在板布局以下控件:只有细胞（未染色的，单独或DAPI罗丹明单独）只和粒子。 <ol><li>开展1-4在无菌细胞培养罩，以防止样品污染的措施都加入试剂。 </li><li>板的巨噬细胞（鼠J774细胞系）在一个96孔板在1-5×10 4个细胞在50μl（每孔）<html>为辅预热媒体。理想板此方法是黑色的96孔板用透明或黑色的塔底，取决于读者的能力。 <ol><li>用于补充培养基，用10％热灭活的FBS，以避免补体级联的干扰，和1％青霉素/链霉素（如果不使用活细菌吞噬作用）。如果使用贴壁细胞允许1-2小时用于粘附，并用于非粘附细胞，降速板与细胞在​​500×g 10分钟。 </li></ol></li><li>在50μl中添加期望的激动剂/拮抗剂或车辆控制在2倍浓度（悬浮于PBS或更优选，在补充培养基）为100微升（1×）的最终体积并孵育时间所需量。多道移液器和试剂水库将有利于最快的处理。 </li></ol> 2.荧光体颗粒的调理作用ftp_upload / 52195 / 52195table1.jpg"WIDTH ="500"/&gt; 表1:测试的细胞类型并在荧光分析荧光颗粒吞噬作用和肌动蛋白聚合的表1示出的细胞类型的组合（细胞系和原代细胞），我们的小组通过荧光法使用。除了看吞噬作用和肌动蛋白聚合由野生型细胞中，我们还利用一些这些颗粒用于通过转染的细胞表达GFP（绿色荧光蛋白）检查吞噬。用含有荧光报告质粒的细胞的荧光，也可以用作除了DAPI 表说明一个细胞标记:A -肌动蛋白聚合; P - 吞噬; PT - 吞噬了GFP标记的转染细胞; OPDex - 调理葡聚糖珠; HKOP - 热杀调理。 <ol><li>确保荧光颗粒和标签避光在任何时候，杜尔为了防止光致漂白，以及在孵育荷兰国际集团的处理，洗涤<ol><li>对于干颗粒，称出荧光热灭活调理（HKOP）菌（E2861: 大肠杆菌 K-12株），使用制造商的规格。小心确保质量占10:1 - 20:1的细菌:细胞比率（或至少10:1的比率-在吞噬量化常用）13,14。可以由制造商提供，或者经由couting为1mg / ml储备液的连续稀释测定颗粒的数目。 </li><li>悬浮在50微升PBS，涡旋在最高设置1分钟。确保适当的涡流（在这里和在整个协议）作为颗粒倾向于聚集可能影响调理作用的功效。 </li><li>为粒子在悬浮液中，如荧光标记的葡聚糖珠（DEX珠），涡流原液1分钟，取50μl的悬浮液，或根据对在卷D的胎圈浓度珠etermine安装方便10:1珠子:细胞比例。 </li></ol></li><li>添加opsonizing试剂等体积的粒子，大力旋涡振荡为一分钟。 </li><li>与opsonizing试剂在37℃下1小时孵育颗粒。 </li><li>继温育后，在500×g离心离心颗粒5分钟，并除去含有过量opsonizing试剂的上清。通过加入100微升的PBS洗涤颗粒，涡旋重悬沉淀，并离心5分钟，在500×g下。 <ol><li>重复这些步骤两次，以确保正确的调理作用，并除去未结合的抗体。 </li></ol></li><li>通过重新悬浮它们的5ml培养基（足够一个96孔板）制备调理粒子的工作溶液，并存储避光直到除细胞。 </li></ol> 3.吞噬<ol><li>放置颗粒在无菌试剂槽的工作溶液。从这里出发，加5056;升调理粒子在步骤1（对于50毫微克/ ml的终浓度）中制备的细胞，并允许细胞吞噬发生在标准细胞培养条件。下面所示是吞噬的两种方法，可使用该方法（ 图2）来评估。 </li></ol><img alt="图2" fo:content-width="4in" src="/files/ftp_upload/52195/52195diagram2.jpg" width="600" /> 图2:连续与同步吞噬比较连续吞噬表示颗粒随时间的连续的内化，因为它们慢慢到达细胞上的井的底部。甲对比同步步骤（离心）迫使粒子到沉底，提高与细胞的颗粒接触，并导致立即的内化的细胞。同步吞噬是一个更快的过程，更迅速内化的一部分icles由于增加的细胞:颗粒接触。 <ol><li><ol><li>对于连续吞噬方法（图2），允许细胞以不断吞噬调理细菌作为它们慢慢沉底和进来与细胞接触时，利用较长的时间帧，以允许所述细菌到达井的底部，并获得在与细胞接触。如果进行一个时间过程实验，在不同时间间隔添加细菌和停止反应的整个板的同时，提高处理速度。 </li><li>对于同步吞噬方法（图2），可提高颗粒:通过离心细胞接触，降速含有细胞和颗粒（5分钟，在500×g离心）只要颗粒加入，以促进所有时间点的同步整板和加速粒子:细胞相互作用。 <ol><li>测量时间过程完成CENTRI后右起fugation一步。如果导通的时间过程实验，如下所述独立地停止各时间点在不同的时间间隔。 </li></ol></li></ol></li></ol>注:同步吞噬方法是与前一个较为费力，但在较短的时间提供了更高的吞噬指数。 4.停止吞噬<ol><li>对于贴壁细胞，除去上清液，并添加50微升稀释台盼蓝的PBS（1:2稀释）中，为了扑灭非内化的细菌的荧光。 </li><li>对于非粘附细胞，降速板并除去上清液。加入50微升稀释台盼蓝。确保到离心机每次洗涤之间的板上于（在500×g离心5-10分钟），以便最大限度地减少细胞的损失和使足够的洗涤。 </li><li>以下锥虫孵育，洗涤细胞2次，用PBS以除去任何残留锥虫（直到PBS除去从井变为清楚）。 TRypan已经显示以猝灭未内在化的荧光标记，热灭活，细菌颗粒的荧光15,16，但不适用于活细菌或OPDex珠。活的细菌，包括使用抗生素，如青霉素，链霉素，庆大霉素或代替的锥虫洗涤，以避免荧光的非内化颗粒混杂影响洗涤。 </li><li>固定的细胞用100μl的4％多聚甲醛，并孵育15分钟，在室温下，然后用PBS（以每孔100μl的每次洗涤）。在此步骤中，保存的细胞在4℃下进行长达一周，或直接进行染色和定量。 注意:所有的后续步骤，形成该点序可以在细胞培养罩的外面来完成。 </li><li>删除所有的液体，并添加50微升的DAPI的重组在PBS 5毫微克/毫升。允许5分钟进行染色，然后用PBS洗两次。在洗涤步骤中，加入50微升PBS至每个孔，并记录吞噬作用。 </l我&gt; </ol> 5.肌动蛋白染色注意:除了吞噬作用，荧光法将使肌动蛋白聚合的量化通过测定肌动蛋白，这是抑制片状伪足，pseudopodiae期间减少的强度，并且膜皱裂，与抑制剂（ 图1A）处理的结果。这是，如果除了吞噬感兴趣的肌动蛋白聚合的量化可选步骤。如果肌动蛋白聚合是最终目标，使用荧光颗粒吞噬作用是可选的。 <ol><li>下列的多聚甲醛固定（ 步骤4.3），用PBS洗涤平板两次，每孔100μl每次洗涤。加50微升0.1％的Triton X-100的PBS透化对孵育在室温-5分钟。 </li><li>用PBS洗出0.1％的Triton X-100的2倍（如上述），并用1％牛血清白蛋白（BSA）20分钟阻断，以避免拉贝的非特异性结合升。 </li><li>封闭后，取出BSA解决方案，并立即加入罗丹明鬼笔。为了制备罗丹明染色的工作溶液中，在5毫升PBS（足够用于一个板）的使用100微升methanoic溶液（由生产商提供）。从工作溶液中添加50微升每孔，并孵育20分钟，在室温下进行。 注:继洗涤步骤和DAPI染色如在步骤4.4的细胞将准备定量。 </li></ol> 6.定量<ol><li>用在λ= 355 nm，发射滤波器激发滤光片在λ= 460nm的量化使用荧光板读数器，记录绿色荧光使用激发滤光片在λ= 488 nm和发射滤波器的吞噬在λ= 5​​18纳米，和蓝色荧光。 </li><li>以通过λ= 355纳米和λ= 460nm的（如上），和红色感量化使用荧光板读数器，记录蓝色荧光的肌动蛋白聚合通过激发滤光片orescence在λ= 5​​84 nm，发射滤波器λ= 620纳米。 注:轨道或中心的检测是可以接受的记录。某些类型的细胞收集更多到阱比中心，在这种情况下，轨道记录可以是优势的边缘。特别是，平均大约轨道三个点也可以被用于获得更准确的结果。 </li><li>划分的绿色或红色荧光由蓝色荧光读数（来自DAPI）总RFU，根据粒子标签或吞噬与肌动蛋白定量（ 图1）。由于高的板到板方差，使用相对指标（如相对于T = 0的控制），以最好地说明观察到的趋势，并促进更可靠的统计分析。 </li></ol>

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The authors have no competing financial interests.

所述荧光技术的主要限制是实验方差以及与广泛洗涤和使用非粘附细胞的细胞相关的损失。 方差很大程度上观察到由于在荧光发色粒子作为制备储液的过程中称重和分注的变化不保持从实验的颗粒进行实验的相同数量的精确方式。为了解决实验之间方差的问题，数据可以表示为相对术语，例如，作为％吞噬作用或折叠从控制变更（控制 - T = 0;加入和除去颗粒立即）。这种形?...

荧光信号的量化已被广泛应用于许多科学的方法，从PCR检测，流式细胞术，激光共聚焦显微镜和FRET分析复ELISA分析。荧光成像和定量有着广泛的应用，并且可针对各种细胞过程的定量分析的一大利器。使用的荧光标记物和它们的信号已经彻底改变了在过去十年中，并且荧光板阅读器的出现促进了荧光期间的细胞过程发出的光的高通量定量。 荧光分析可以作为吞噬量化一个伟大的工具。由于吞噬细胞的发现通过Metchnikoff在1800 1吞噬了研究。多年来，各种方法已被用于研究这个重要过程抵御入侵细菌，病毒，真菌和寄生虫病原体2-5先天免疫防御必需。量化利用显微镜和体视技术，以以前的方法以可视化的细胞被吞噬，然后将其通过计算内化颗粒（手动或使用利用软件）6-8的定量。一些缺点单独使用显微镜进行定量分析是细菌人工计数是劳动密集，更容易观察偏见。在吞噬的量化使用的另一种方法是从细胞裂解物（以下吞噬）上的细菌培养板细菌电镀的微生物技术，但这种方法可能会失败占杀菌机制和部分内在的细菌的存在。相比显微镜此方法甚至更多的劳动密集和需要几天的时间来分析。流式细胞术似乎是量化吞噬的最快，最有效的方法，并已用于通过许多组9-11，但成本高，通常与在相关联的所需的分析仪器使得它的最昂贵的方法时，相比于前面提到的测定。 该荧光法是一个很好的替代流式细胞仪粒子内部的分析，因为它提供了用荧光设备偏见的量化，是不是因为成本太高。荧光的其它额外的好处是效率高，高吞吐量能力用于定量荧光的标记的内化的颗粒排出。 荧光的好处是可以外推到比吞噬其它过程定量。例如，荧光分析，可用于研究任何导致肌动蛋白聚合的变化在细胞内或膜结合的受体，改变细胞通透性/生存力，转染效率，以及调制的表达过程。荧光技术的一个缺点是，根据所使用的标签，可能有一个高实验实验变异性可通常通过证明通过相对定量数据，如倍数变化或从控制增加的百分比来解决。

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			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:131px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:315px;"></td>
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			<td style="width:255px;"></td>
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			<td height="66" style="height:66px;width:315px;">1-&#956;m yellow-green fluorescent Fluo-Spheres;</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">F8852</td>
			<td style="width:255px;">combine 50&#956;l with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
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			<td height="87" style="height:87px;">Heat killed E. coli BioParticles fluorescein conjugate</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2861</td>
			<td style="width:255px;">reconstitute (5 mg) in 50&#956;l of PBS and combine with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Opsonizing reagent</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2870</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rhodamine phalloidin</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">R415</td>
			<td style="width:255px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Trypan blue</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;">15250-061</td>
			<td style="width:255px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
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			<td height="21" style="height:21px;width:315px;"></td>
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		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">the items below are available in many brands but the items we used in this study are from the following manufacturers</td>
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		<tr height="66">
			<td height="66" style="height:66px;width:315px;">RPMI Cell growth media&#160;</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">Supplemented with 10% FBS and 1% pennicillin/Streptomycin; warm in 37oC before use</td>
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			<td height="21" style="height:21px;width:315px;">Fluorescent plate reader-Fluostar Omega</td>
			<td style="width:131px;">BMG Labtech</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
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			<td height="21" style="height:21px;width:315px;">Paraformaldehyde (16%)</td>
			<td style="width:131px;">Fischer Scientific</td>
			<td>AA433689M</td>
			<td style="width:255px;">dilute to 4% before use</td>
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			<td height="42" style="height:42px;width:315px;">96 well plates</td>
			<td style="width:131px;">Greiner</td>
			<td style="width:119px;">655097</td>
			<td style="width:255px;">clear or black or clear bottom - black plates</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Multichannel pipette (8-12 channels)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Reagent reservoirs</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1x PBS</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microfuge tubes (0.6 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">&#160;Conicles (10 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
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high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization

荧光分析的目的是作为一种有效的，成本有效的，高通量分析的吞噬作用和其他细胞过程的方法。这种技术可以用在各种细胞类型，包括粘附和非粘附的，以检查各种细胞特性。当研究吞噬作用，荧光技术利用吞噬细胞类型如巨噬细胞，和荧光标记的调理粒子的荧光可以以台盼蓝的存在而熄灭。下面粘附的巨噬细胞的电镀在96孔板中，荧光发色粒子（绿或红）施用和允许细胞吞噬对于不同量的时间。以下荧光体颗粒的内在化，细胞用台盼蓝，这有利于荧光信号的消光从其中不内化，或仅仅是附着于细胞表面的细菌。继锥虫洗涤，将细胞用PBS洗涤，固定，一次用DAPI染色（蓝色核荧光标记），其用于标记细胞的细胞核。通过板的核（蓝）读取或颗粒的简单荧光定量（红/绿）荧光我们可以检查的绿色相对荧光单位的比例:蓝色和确定一个表示每个细胞内在荧光的细菌量的吞噬指数。测定使用96孔的方法和多道移液器用于洗涤的持续时间，从吞噬数据采集结束的结束，是小于45分钟。流式细胞术以类似的方式也可以使用，但荧光的优点是它的高通量，评价与样品最小操纵和每个细胞的荧光强度的快速定量快速方法。类似的策略可以应用到非粘附细胞，活标记的细菌，肌动蛋白聚合，并且基本上任何利用荧光过程。因此，荧光是一种有前途的方法，其成本低，高throughpUT能力的细胞过程的研究。

通过使用该协议的量化吞噬作用和随后的肌动蛋白聚合的两种主要方法是观察连续或同步吞噬作用。 显微镜分析（ 图1B）示出了荧光图像媲美什么荧光正在记录（也参见图1）。在图1B是红色荧光的肌动蛋白，绿色荧光268 FITC的标签HKOP E.大肠杆菌 ，核DAPI染色，三个人269的合并后的图像荧光团在一起。这个图像显示有效的吞噬作用，...

Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

high-throughput-fluorometric-technique-for-assessment-macrophage

Research

JoVE Journal

Immunology and Infection

14.6K 次观看.  University of Minnesota. Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

视频: High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

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, ...

科研

免疫与感染

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

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

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

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

巨噬细胞胆固醇消耗及其对的吞噬作用<em&gt;新型隐球菌</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

肺泡巨噬细胞吞噬作用与小鼠细菌清除

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...