我们感谢的皇家阿尔弗雷德王子“和”儿童权利公约“和正常的肠道黏膜的新鲜样品收集的协和遣返医院解剖病理学实验室的工作人员。这项工作是由癌症研究所的新南威尔士转化项目资助资助。

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="图1" /> 图1。暂停从手术的CRC样本活体细胞的准备工作流程。 1。临床样本分类所有样品均采自皇家阿尔弗雷德王子医院（坎珀，NSW，澳大利亚）和协和遣返医院（协和西澳大利亚新南威尔士州）X08 - 164号议定书下的知情同意。 <ol><li>收集的新鲜大肠癌（CRC）的或腺瘤标本，和正常的肠道黏膜至少10厘米的肿瘤。汉克的平衡盐溶液的pH值7.3（HBSS）在4 ° C储存长达12 h切除后的样品。 </li><li>按照人类病原体的安全法规，在II级生物安全柜过程中所有的临床样本。剖析到2毫米的多维数据集使用两个手术刀片在培养皿菜的样品。 </li><li>孵育肿瘤和正常组织，在单独的Eppendorf管，偶尔温柔的搅拌60分钟，在37 ° C与同等体积的RPMI 1640培养基含2％（V / V）型胶原酶4（沃辛顿，翠湖，美国新泽西州）和0.1 ％（W / V）从牛胰腺中的脱氧核糖核酸我（DNA酶I，Sigma - Aldrich公司）。 </li><li>部队半消化组织通过精细的丝网，用10毫升注射器的柱塞滤网，通过与HBSS洗涤细胞。 </li><li>传递导致细胞悬液通过200微米和50微米Filcon的过滤器（BD Biosciences公司），以去除细胞聚集。大多数被删除的DNA，粘液和细胞聚集在这个系列的过滤。 </li><li>离心细胞悬浮液在20 ° 5分钟的400 XG。 </li><li>热灭活的FCS悬浮细胞沉淀，用含10％二甲基亚砜（DMSO），慢慢地冻结在离心管和储存在-80 °。冻结过程中往往会降低样本中的粘液和裂解红细胞。 </li></ol> 2。细胞采集的样品制备<ol><li>解冻样品很快在37度水浴和10毫升的HBSS洗出二甲基亚砜悬浮细胞。 </li><li>离心细胞悬浮液在20 ° 5分钟的410 XG。 </li><li>弃上清，重悬细胞沉淀在500μL的HBSS中。 </li><li> 0.1％（W / V）DNA酶在室温下20分钟我的样本对待。 </li><li>台盼蓝等体积混合每个细胞悬液10μL，10μL的混合物装入一个血球。使用光学显微镜100倍的放大倍数，计数活细胞，出现明确的台盼蓝拒，死细胞染料。最低4 × 10 6活菌细胞上的芯片捕获。 </li><li> DNA酶处理后，重新悬浮在10毫升的HBSS和离心机的细胞悬液，在20 ° 5分钟在410 XG。 </li><li>倒出到终体积为200μL的RPMI 1640培养上清和悬浮细胞沉淀。 </li></ol> 3。抗体芯片的细胞捕捉<ol><li>浸湿浸入磷酸盐硝化棉节DotScan抗体芯片缓冲液（PBS约20秒）仔细擦拭折叠Kimwipes芯片的玻璃边缘，避免用手触摸硝酸纤维素部分。 </li><li>加水至芯片孵化托盘提供湿热试验箱。进入会议厅的芯片和潮湿的硝化棉部分滴入的RPMI 1640细胞悬液。移液器滴硝化棉的每个角落，以确保即使细胞扩散。 </li><li>在37 ° C孵育1小时的芯片孵化允许细胞定居和接触芯片上的抗体。细胞中表达表面抗原相应的抗体，他们的土地上，将被捕获。 </li><li>孵化后，轻轻沾的芯片和垂直方向上分为三个载有至少15毫升PBS洗掉未结合的细胞（每洗20秒）的波谷。 </li><li>准备3.7％（W / V）甲醛的PBS，修复细胞和抗体的交联。轻轻吸管约1毫升，包括硝化棉的芯片部分。孵育20分钟在室温。 </li><li>下一步DIP芯片的PBS（15毫升; 30秒）的变化分为3个洗出多余的甲醛。 </li><li>擦拭边缘和背面与Kimwipes载玻片和使用的DotScan扫描仪，同时扫描芯片，硝酸纤维素部分是湿润的。光学扫描提供例如CRC混合细胞群的细胞，白细胞和其他间质细胞的肿瘤抗原的表达模式。 </li></ol> 4。荧光复<ol><li>从扫描仪中取出芯片适用于200μL封闭液（2％W / V BSA，2％热灭活的人AB血清，PBS，pH值7.3）。在室温孵育20分钟的芯片托盘。 </li><li>准备的multip词法用铝箔覆盖的Eppendorf管中的解决方案：20μL藻红蛋白 - 抗CD3（Beckman Coulter公司，Gladesville，NSW，澳大利亚，＃IM12824; 1/7.5最终稀释），10μL的Alexa Fluor 647的抗EpCAM的（Biolegend美国加利福尼亚州圣迭戈的1 / 15稀释），2μL热灭活的人AB血清（Sigma - Aldrich公司，城堡山，新南威尔士州，澳大利亚）和118μL封闭液。 </li><li>从微阵列流掉多余的封闭液，滴入硝酸纤维素部分复用的解决方案，散布均匀。为在黑暗中30分钟，在室温下孵育。 </li><li> DIP芯片;纵向分为三个波谷15毫升新鲜PBS（每30秒）。 </li><li>让干的芯片，在黑暗中和存储在4 ° C在幻灯片框。该芯片可存储长达3个月没有损失的荧光，在黑暗中。 </li><li>扫描使用分辨率设置为50台风FLA 9000扫描仪（GE医疗，Rydalmere，NSW，澳大利亚）（532 nm激光，580 BP30 PE排放过滤器干燥的芯片。633 nm激光Alexa的647和670 BP30排放过滤器）。微阵列扫描硝化棉方面临在玻璃板上扫描仪托盘向下。 </li><li>保存为TIFF文件的荧光图像，使用Photoshop设置图像的大小为17 x 25厘米，分辨率72像素/厘米。 DotScan分析软件导入图像分析的点的强度。 </li><li> DotReader捕捉一个点结合模式的数字图像和量化每个抗体点的密度，一个8位的灰度范围（1-256 ü）的细胞结合。偶尔非特异性亚型控制绑定是绑定值中减去相应的免疫球蛋白亚型的抗体。对100％的强度设置最亮的点，每个点的荧光强度芯片正常化。在一个XML文件（原始数据）记录信号强度/现货或条形图中的一个代表。PDF文件（最终报告） </li><li>芯片heatsmaps和分层聚类进行使用TM4芯片软件套装（MultiExperiment的浏览器（兆电子伏） <a href="http://www.tm4.org/mev.html">版本</a> 4.4 http://www.tm4.org/mev.html ）。分层聚类进行调整的背景，使用完整的连锁分析MeV的数据。欧氏距离的相似性度量。 2尾的学生等方差t -检验来确定结果的统计学意义。 </li></ol> 5。代表性的成果： 从DotScan芯片的结果应该显示一致的细胞之间的重复阵列的结合模式。有力契合点绑定（CD44/CD29）使一个被放置在数组区域电网。图2给出了优化细胞的捕获和复用的一个例子。图3显示了细胞的捕获和可能的解决方案过程中遇到的一些常见问题。 芯片细胞结合的结果是可以量化的测量点上的灰度规模从1到256不等表达强度。图4显示了数值数据，从58手术的CRC样本，EpCAM的Alexa的647抗体染色，作为一个层次聚类的热图。即使样品的数量是有限的的，同一阶段的CRC往往在同一组群。 <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="图2" /> 图2（澳大利亚临床病理分期，机场核心计划的阶段B1）大肠癌的临床肿瘤细胞结合模式。 （一）DotScan抗体键重复的微阵列（概述）的左半边显示抗体地点。顶部包含DotScan白血病芯片原有的82抗体。额外的40抗体，对应于特定的表面抗原发现在文献中被上调，作为一个CRC“卫星”芯片的补充。下面的部分，由同型对照抗体（二）“儿童权利公约”细胞​​结合微阵列的光学图像。 （C）CD3 + T细胞荧光图像显示。 （四）EpCAM的荧光图像显示的CRC细胞。 <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="图3" /> 图3。贫困DotScan结果和可能的解决方案的例子。 （一）低细胞结合的解决方案：确保至少4x106可行的细胞阵列上（二）同型控制约束性和非特定的细胞结合;解决方案：孵化前对芯片的热灭活人AB血清样品，以尽量减少同型对照具有约束力。有时，硝化纤维细胞的非特异性约束力的少量发生与CRC的样品，并没有显著影响的结果。 （三）干燥硝化棉孵化过程中，解决方案：确保样本覆盖了整个的硝化棉部分和芯片是在一个平面上培养。 （四）高背景工件;解决方案：确保芯片是彻底清洗下孵化。 <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="图4" /> 图4。DotScan分析软件生成的柱状图代表细胞结合上灰度规模的密度范围从1到256。轴的数字是指CD抗原。其它缩写的TCR，T细胞受体;κ，λ，免疫球蛋白轻链; SIG，表面免疫球蛋白; DCC，在大肠癌蛋白删除;表皮生长因子受体，表皮生长因子受体; FAP的，成纤维细胞激活蛋白的HLA - A，B， HLA - DR的彗星，人类白细胞抗原DR和A，B，C分别;云母，MHC I类链相关蛋白; MMP - 14，矩阵metallopeptidase 14; PIGR，聚免疫球蛋白受体; TSP - 1，凝血酶- 1;美罗华，源化抗CD20。 <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">点击这里查看大图</a> 。

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没有利益冲突的声明。

在这段视频中，我们将演示如何DotScan抗体芯片可以在一个简单的，半定量的方法来研究从启联资源中心组织的细胞群表面抗原型材的使用。 从组织中获得一个可行的单细胞悬液，实验的成功是至关重要的，因为似乎是公司具有约束力的全细胞抗体点的孵化过程中所需的能源依赖的过程（如抗原上限和/或伪足形成），而随后冲关死细胞。 1型胶原酶最初受雇于组织disaggregation8?...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th>试剂或设备名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>汉克斯“平衡盐溶液</td><td> Sigma - Aldrich公司</td><td> H6136 - 10X1L </td><td>缓冲25毫米HEPES（Sigma公司＃H3375） </td></tr><tr><td> Airpure生物安全柜II类</td><td>西屋电气公司</td><td> 1687-2340/612 </td><td></td></tr><tr><td>手术刀</td><td>利文斯通</td><td> 090609 </td><td>每包100 </td></tr><tr><td> 2毫米HEPES的RPMI 1640 </td><td> Sigma - Aldrich公司</td><td> R4130 - 10X1L </td><td></td></tr><tr><td>型胶原酶4 </td><td>沃辛顿</td><td> 4188 </td><td></td></tr><tr><td>脱氧核糖核酸1 </td><td> Sigma - Aldrich公司</td><td> DN25 - 1G </td><td></td></tr><tr><td> Terumo公司注射器（10毫升） </td><td> Terumo公司</td><td> SS 10大号</td><td>盒：100个</td></tr><tr><td> Filcon过滤器（200微米） </td><td> BD公司</td><td> 340615 </td><td></td></tr><tr><td> Filcon过滤器（50微米） </td><td> Filcon过滤器（50微米） </td><td> Filcon Filcon过滤器（50微米）过滤器（50微米）340603 </td><td></td></tr><tr><td>胎牛血清</td><td> GIBCO / Invitrogen公司</td><td> 10099-141 </td><td></td></tr><tr><td>离心5810 ř </td><td> Eppendorf公司</td><td> 7017 </td><td></td></tr><tr><td>二甲基亚砜</td><td> Sigma - Aldrich公司</td><td> D2650 </td><td></td></tr><tr><td>台盼蓝</td><td> Sigma - Aldrich公司</td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td>赫斯曼</td><td>不可用</td><td></td></tr><tr><td>光镜下</td><td>尼康</td><td>尼康TMS </td><td></td></tr><tr><td> Cyrovial管</td><td>格雷纳生物一</td><td> 121278 </td><td></td></tr><tr><td>低温冷冻contrainer </td><td> NALGENE </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan抗体芯片试剂盒</td><td> Medsaic </td><td>不可用</td><td></td></tr><tr><td> DotScan芯片洗托盘</td><td> Medsaic </td><td>不可用</td><td></td></tr><tr><td> KimWipes </td><td>金佰利</td><td> 4103 </td><td></td></tr><tr><td>甲醛37％ </td><td> Sigma - Aldrich公司</td><td> F1635 - 500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td>不可用</td><td></td></tr><tr><td>牛血清白蛋白</td><td> Sigma - Aldrich公司</td><td> A9418 - 10G </td><td></td></tr><tr><td>热灭活AB血清2％ </td><td> Invitrogen公司</td><td> 34005100 </td><td></td></tr><tr><td>藻红蛋白的共轭CD3 </td><td> Beckman Coulter公司</td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 -共轭EpCAM的</td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td>台风的FLA 9000 </td><td> GE医疗集团</td><td> 28-9558-08 </td><td> 532 nm激光，580 BP30 PE排放过滤器。 633 nm激光和670 BP30 Alexa647排放过滤器</td></tr><tr><td> MultiExperiment浏览器V4.4 </td><td> TM4芯片软件套件</td><td>打开 - 源码软件（参11） </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

依靠目前的预后和“儿童权利公约”的分类分期，病理组织学和临床研究结果结合起来的系统。然而，在大多数“儿童权利公约”的情况下，细胞功能障碍是众多的基因突变，修改蛋白表达和翻译后修饰1的结果。 一些细胞表面抗原，包括集群分化（CD）抗原，已被确定为潜在的预后或转移的生物标志物在CRC。这些抗原的表达理想的生物标志物往往与肿瘤进展或与其他类型的细胞，如互动的变化的肿瘤浸润淋巴细胞（TILS）和肿瘤相关的（TAMS）巨噬细胞。 使用免疫组织化学（IHC）癌症的子分类和预测是公认的对某些类型的肿瘤2,3。但是，没有一个“标记”显示预后意义大于临床病理分期或获得广泛接受，在所有儿童权利公约“情况下的常规病理报告为使用。 最近的方针疾病表型的预后分层依赖的表面蛋白的配置文件使用多个“标记”。虽然表达的肿瘤蛋白质组技术如iTRAQ分析是一个功能强大的工具biomarkers4发现，它不是诊断实验室常规使用的最佳选择，不能区分不同类型的细胞在混合人口。此外，肿瘤组织大量的纯化的质膜糖蛋白的分析，这些方法都需要。 在这段视频中，我们描述了一个简单的方法，从分类的CRC使用DotScan CRC抗体芯片的样品活细胞的表面蛋白质组分析。 122抗体芯片包括一个标准的82 -抗体确认系特定标记的白细胞粘附分子，受体和炎症和免疫反应 5标志物的范围，CRC的40个潜在的预后标志物的检测与卫星地区的区域。细胞被捕获，只对他们表达相应抗原的抗体。光学扫描确定，每个点的细胞密度，反映了细胞表达，抗原，抗原的表达和亲和力的抗体6水平的比例。 CRC的组织或正常的肠道黏膜，光学扫描反映免疫细胞混合种群。然后，可以使用复用荧光来分析细胞阵列上捕获的利益选择的细分人群。例如，Alexa的647抗上皮细胞粘附分子（EpCAM的; CD326），是一个泛上皮被用来检测的CRC细胞和正常的肠道黏膜上皮细胞的分化抗原，而藻红蛋白 - 抗CD3，检测浸润T细胞7。 DotScan的CRC芯片应诊断替代的解剖学基础CRC分期系统的原型。

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

我们描述了一个过程分解大肠直肠癌（CRC）来产生可行的单个细胞，然后在定制的抗体，认识表面抗原（DotScan CRC芯片）芯片捕获。绑定到芯片的细胞亚群可以分析使用荧光标记的单克隆抗体与荧光染料的复用。

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K 次观看.  University of Sydney. 我们描述了一个过程分解大肠直肠癌（CRC）来产生可行的单个细胞，然后在定制的抗体，认识表面抗原（DotScan CRC芯片）芯片捕获。绑定到芯片的细胞亚群可以分析使用荧光标记的单克隆抗体与荧光染料的复用。

视频: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

钩端螺旋体暴露于表面的蛋白免​​疫荧光法

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

抗体标记用荧光染料使用磁蛋白A和G蛋白珠

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

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.