在此发表研究报告由美国国家癌症研究所的国家机构的健康奖号码R01CA174385下的支持。的内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1。试剂准备<ol><li>准备通过混合500毫升的RPMI-1640和5毫升的RPMI-PLGH每个青霉素 - 链霉素，L-谷氨酰胺，和HEPES溶液。 </li><li>在混合用10％胎牛血清（FBS）的RPMI-PLGH准备R10溶液。 FBS是于56℃下30分钟，加入之前热灭活。 </li><li>预保温在37℃下50毫升的RPMI-PLGH的，15毫升PBS中，和15ml无菌锥形管中的R10。 </li><li>在PDMS：硅主阵列的nanowells采用光刻基本上如前所述，10的制造。调匀Sylgard的184弹性体套件碱剂和固化剂按10:1的重量比在一个一次性杯使用塑料刀。脱气的混合物1小时，在真空室中。到纳为阵列，倒入密封载玻片上，让它坐在30分钟。的组件转移到设置为80℃下进行2小时的烘箱中，并在室温下搅拌1小时，放凉。弹性体电流E在此期间，将债券载玻片上。含有PDMS纳为阵列的载玻片仔细升空硅主站，覆盖着透明胶带和保存，直到将来使用。 </li></ol> 2。靶细胞（T）标签<ol><li>使用血细胞计数器和颗粒细胞计数5000000从细胞培养的靶细胞在无菌的15毫升锥形管，通过离心分离，在340×g离心5分钟。被分裂的细胞培养的前一天，在5毫升的总体积（密度= 1万个/ ml），并培养在T-25烧瓶（VWR）。已经描述了用于细胞计数使用血细胞计数仪的协议在前面已经详细11 </li><li>吸出多余的介质，留下了约50微升的媒体。 </li><li>准备一个有效的解决方案的细胞追踪红（CTR）的混合0.5微升CTR股票（1毫米），150μLRPMI-PLGH（终浓度为2.5μM）。到靶细胞的工作溶液，并加入150μl调匀使用P200的吸移管。PKH 26（最终浓度为2μM）或者，可以使用的标签的靶细胞，按照制造商的说明。 </li><li>在37℃/ 5％CO 2中孵育20分钟。 </li></ol> 3。效应（E）细胞标记<ol><li>计数5000000效应细胞在无菌的15毫升锥形管使用血细胞计数器和颗粒细胞在340×g离心5分钟，通过离心从细胞培养。 </li><li>吸出多余的介质</li><li>准备Vybrant DyeCycleViolet染色溶液的工作溶液的1微升的5mM Vybrant紫罗兰库存通过添加1毫升的R10培养基中（终浓度为5μM）。添加到细胞中的工作溶液，并重新悬浮使用P200的吸移管。 PKH 67（最终浓度为2μM）或者，可以使用的标签的靶细胞，按照制造商的说明。如果使用PKH 67，然后SYTOX格林不应使用，因为它们是在相同的荧光信道。凋亡的目标是只使用一个枚举在这种情况下，nnexinV染色。 注：如果执行的时间推移，而不是端点测量，应避免UV染料类似Vybrant紫罗兰。 </li><li>在37℃/ 5％CO 2中孵育20分钟。 </li></ol> 4。使用密度梯度活的和死细胞的分离<ol><li>加入6.8毫升和6.0毫升RPMI-PLGH的目标和效应细胞，分别由上下吹打轻轻混合。 </li><li>层的FICOLL帕克加上每个锥形管的底部包含目标和效应细胞的3.5毫升。层FICOLL慢慢地确保形成层之间FICOLL和媒体。 </li><li>在340×g离心30分钟，离心两管。没有制动和加速。 </li><li>在离心过程中，转移到4 - 孔板7毫升预先温热的PBS。准备纳为阵列（PDMS中制作）：清洁底部的载玻片上，用乙醇和氧化PDMS在高RF设置使用1分钟的等离子体氧化。这一步将STErilize纳为阵列中，同时也使PDMS的亲水性。立即将纳为阵列到板PBS。 </li><li>板，生物安全柜，吸去多余的PBS。 10毫升1％高尚的琼脂100毫克琼脂粉加入10毫升去离子水，然后微波30-45秒。应用温暖1％贵金属琼脂上的顶部和底部边缘的载玻片，包含纳为阵列固定阵列，等待10分钟的琼脂固化。加入3毫升PBS和吸出多余的琼脂。 </li><li>加入3毫升R10纳为阵列的顶部上，并让它〜10分钟的平衡。 </li><li> FICOLL离心分离之后，从每个管顶端层吸5毫升媒体。要小心，不要吸出含有细胞层。 </li><li>收获细胞（白层）通过回收从与P1，000吸移管和移动到新的无菌锥形管FICOLL和介质层之间的1-2毫升。重复此为套细胞，同时确保你的格言IZE恢复细胞。 </li><li>加入3毫升预热的RPMI-PLGH每个管中，通过移液混合和颗粒通过离心分离在340×g离心5分钟，充分的加速度和制动。 </li><li>吸出多余的介质，加入5毫升预热的RPMI-PLGH，每个试管和，重复离心340×g离心5分钟。 </li><li>吸出多余的介质和悬浮细胞沉淀在500μl的R10。 </li><li>使用台盼蓝排除方法血球计数器的计数活细胞。 </li></ol> 5。手机装上纳为阵列<ol><li>吸纳为阵列的多余的介质，并加入2毫升的新鲜R10在阵列中。吸再次而确保纳为阵列的顶部是不是太湿/干的，因为它会影响细胞分布。 </li><li>存款1×10 5效应细胞纳为阵列表面在200微升R10到（密度= 5×10 5细胞/ ml）。 </li><li>让细胞沉降5分钟，并使用标准的组织培养显微镜检查为en确定细胞的分布优选。如果需要添加更多的细胞。 </li><li>存款1×10 5靶细胞在200μlR10纳为阵列表面上（密度= 5×10 5细胞/ ml）。 </li><li>让细胞5分钟，并使用标准的组织培养显微镜检查，以确保细胞的分布优选定居。如果需要添加更多的细胞。 </li><li>仔细冲洗纳为阵列，用2毫升R10和吸出多余的介质。 </li><li>准备在15毫升无菌锥形管3毫升预先温热的R10。加入3μl的0.5mM的SYTOX格林核酸着色漆（最终浓度为0.5μM）和60μl的膜联蛋白V-的Alexa Fluor 647准备工作溶液。仔细，同时确保细胞不从井到4孔板中添加有效的解决方案。 </li><li>在37℃/ 5％CO 2中孵育15分钟。 </li></ol> 6。成像1 <ol><li>采集图像的纳为阵列使用荧光MICR的Oscope：在这个例子中，我们使用ZEISS观察，Z-1显微镜配备了一个机动阶段和Lambda DG4。 </li><li>初始化的软件和显微镜检查的信号强度上的透射光，和其他荧光通道。总体而言，将有五道相对应的透射光，膜联蛋白V-647（Cy5的过滤器），CTR（红色荧光蛋白过滤器），SYTOX绿色（FITC过滤器），并Vybrant的紫（DAPI过滤器）。确保显微镜的设置，以确保最大的信号噪音，同时避免饱和。 </li><li>设置为纳为阵列的X和Y位置。零位置设置和初始化焦点上的中心的7×7纳为阵列块上的纳为阵列邮票左上角开始过程。设置的x，y的位置，以配合每个块的中心的z值，以准确地反映在每个块上的焦点。 </li><li>一旦位置和焦点都设置，确保线性模式设置图像采集和采集图像。 <s豪华型&gt;注意：如果需要打开孵化器和CO 2在显微镜实验前至少30分钟。 </li></ol> 7。成像后转移到6小时的孵化器（37℃/ 5％CO 2）的4 -孔板含有纳为阵列。 8。成像2 采集图像在所述第二时间点，在步骤6中所概述。 9。图像分析<ol><li>细胞内的nanowells占用确定通过使用适当的图像分割程序（ 例如 ImageJ的， <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ）基本上如前面所述的9,12。 </li><li>的显微镜图像（t = 0时小时; 吨0）的初始集合的分析是用来生成一个数据库含有一个活的靶细胞（确定个别nanowells[的CellTracker红色，红色），无死细胞（Annexin V染色鉴定，绿色）[T1T 0 nanowells。 </li><li>显微镜图像的第二组（ 吨 = 6小时;吨6）分析，用于独立地确定一个第二数据库的nanowells含有1靶细胞（红色）[T1T 6 nanowells]。实现的完整性校验（数据库查询），以确保在t = 0的所有活靶在t 6的目标相匹配，以产生最终的集的匹配目标（作为具有T1T 0 = 6 T1T指定为T1match的nanowells实现） </li><li>在nanowells效应细胞的数目被确定使用Vybrant紫染色在 t 0 [E1 nanowells] </li><li>实现数据库查询确定含有单效应的nanowells共同培养的单一目标（E1和T1的比赛作为nanowells，指定为E1T1的定义） </li><li>由我打进效应诱导细胞凋亡<html> dentifying的目标是在t = 0和Annexin V阳性T 4（E1T1与Annexin V的目标染色在t 6），膜联蛋白V负。 </li></ol> 10。可选的延时成像的杀戮使用尼康BioStation IM <ol><li>盖玻片底培养皿中切出一小片的纳为阵列芯片（步骤5）编制的盖玻片上，将完全适合。请确保该芯片切割时，保持湿润。 </li><li>混合料1×10 6个目标和1×10 6的T细胞在100μl培养基，移液到盖玻片。 </li><li>允许约15-30分钟，让细胞定居。 </li><li>快速反转的小纳为阵列芯片，并把它放在盖玻片。 </li><li>为了确保芯片将无法移动的摄像过程中，放置芯片的顶部上的一个或两个20毫米x 20mm的盖玻片。轻轻地施加压力，推动芯片的底部的菜。 </li></ol>10吨“&gt; 注：的纳为阵列芯片的底部是过于厚的高清晰度成像，因此具有向被打开倒挂，在此期间，许多细胞被洗出。由于本可以不被容易控制，细胞数步9.2在步骤9.3中，孵育时间必须进行优化设计。

<ol>
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没有利益冲突的声明。

我们概述了协议，用于高吞吐量的单细胞的细胞溶解测定启动阵列nanowells（ 图1）中的效应和目标通过共同孵育。除了吞吐量的技术的一个主要优点是能够监视对所希望的靶细胞效应的介导的细胞毒性，而不需要为这又允许使用自体或匹配/主肿瘤细胞作为靶细胞的靶细胞工程。的空间限制允许检索并随后在结束测定9功能性T细胞克隆。检测的一个限制是需要专门的自动化阶段?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> RPMI-1640 W / O型L-谷氨酰胺</td><td> Cellgro </td><td> 15-040-CV </td><td></td></tr><tr><td>青霉素，链霉素</td><td> Cellgro </td><td> 30-002-CI </td><td> 10,000 IU青霉素10,000μg/ ml的链霉素</td></tr><tr><td> L-谷氨酰胺</td><td> Cellgro </td><td> 25-005-CI </td><td> 200毫米的解决方案</td></tr><tr><td> HEPES </td><td> Sigma Aldrich公司</td><td> H3537 </td><td> 1M </td></tr><tr><td>牛胎儿血清（FBS）的</td><td>亚特兰大生物</td><td> S11150 </td><td>地段测试</td></tr><tr><td>手机追踪器红染色</td><td> Invitrogen公司</td><td> C34552 </td><td> 50微克</td></tr><tr><td> Vybrant DyeCycle紫染色</td><td> Invitrogen公司</td><td> V35003 </td><td> 5毫米</td></tr><tr><td> SYTOX绿色核酸染料</td><td> Invitrogen公司</td><td> S7020 </td><td> 5毫米</td></tr><tr><td>膜联蛋白V-的Alexa Fluor 647 </td><td> Invitrogen公司</td><td> A23204 </td><td> 500μL </td></tr><tr><td>的Dulbecco PBS </td><td> Cellgro </td><td> 21-031-CV </td><td> 500毫升</td></tr><tr><td>高贵的琼脂</td><td> DIFCO </td><td> 2M220 </td><td> 100克</td></tr><tr><td>台盼蓝</td><td> Sigma Aldrich公司</td><td> T8154 </td><td> 0.4％液，无菌过滤</td></tr><tr><td>血球计数仪</td><td>豪塞尔科学</td><td> 1492 </td><td>亮线</td></tr><tr><td> 4 - 孔板</td><td>赛默飞世尔</td><td> 167603 </td><td></td></tr><tr><td> Harrick旅游等离子清洗机</td><td> Harrick旅游等离子</td><td> PDC-32G </td><td>基本的等离子清洗机</td></tr><tr><td> Observer.Z1 </td><td> ZEISS </td><tD&gt; </td><td>荧光显微镜（与下面的三个部分） </td></tr><tr><td>拉姆达10-3 </td><td>萨特仪器</td><td></td><td>过滤器控制器</td></tr><tr><td>拉姆达DG-4 </td><td>萨特仪器</td><td></td><td>超高速波长转换器</td></tr><tr><td>滨松EM-CCD相机</td><td>滨松</td><td> C9100-13 </td><td> CCD显微镜摄像头</td></tr><tr><td> 15毫升锥形管</td><td> BD猎鹰</td><td> 352097 </td><td></td></tr><tr><td> 50毫升锥形管</td><td> VWR </td><td> 3282-345-300 </td><td></td></tr><tr><td>尼康Biostation </td><td>尼康仪器公司</td><td> Biostation IM </td><td></td></tr><tr><td>玻璃底培养皿</td><td> MatTek公司</td><td> P35G-0 </td><td> 35毫米培养皿中，10毫米微孔</td></tr></tbody></table>

quantitative high-throughput single-cell cytotoxicity assay for t cells

肿瘤免疫治疗可以利用的目标和消灭肿瘤的特异性免疫反应。继细胞治疗（ACT）T细胞过继的转基因表达的嵌合抗原受体（CAR）的基础上，已表现出相当大的希望在临床试验中1-4。有几个好处使用CAR + T细胞治疗癌症的能力为目标，非MHC限制性抗原和功能化的T细胞以达到最佳的生存，归巢内的主机和持久性，并最终诱导细胞凋亡的CAR + T细胞在宿主毒性5的事件。 划定的最佳功能的CAR +与临床受益相关的T细胞是必不可少的设计的临床试验的下一代。多光子显微镜等活的动物成像的最新进展，彻底改变了我的免疫细胞功能的研究体内6,7。虽然这些研究已经推进了我们理解，T细胞在体内的功能，基于T-细胞的ACT在临床试验中需要需要链接分子和功能特性的T-细胞的准备预浸液与临床疗效输注后，通过利用在体外实验监测T细胞的功能，如，细胞毒性和细胞因子分泌。已经开发了确定在单细胞水平的T细胞数量的整体运作，但这些是不适合用于监测共轭形成和寿命或相同的小区的能力，杀多个目标8标准基于流式细胞仪检测。 微型阵列设计生物相容性的聚合物，如聚二甲基硅氧烷（PDMS）是一个特别有吸引力的方法，在空间上限制小体积效应和目标9。结合时间的推移自动荧光显微镜，寿usands效靶相互作用可同时监控纳为阵列的成像个别井。在这里，我们提出一种高通量的方法，用于监测在单细胞水平，可以广泛地应用于研究T细胞的细胞溶解功能的T细胞介导的​​细胞毒性。

在图2中的应用程序的一个示例的高通量的细胞溶解测定表明。简要地说，标记的CD19-特异性CAR + T细胞的共同培养与标记的小鼠EL4靶细胞中的纳为阵列的各个井（第1-5）。最初的图像被记录在自动荧光显微镜来识别占用阵列（第6章）（效应和/或目标）的每一个纳为。用于图像处理，找出所有包含一个单一的目标和单效应和nanowells以排除nanowells含有死的目标（第9条）。被转移?...

Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

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

我们描述了一种单细胞的高通量的检测，以测量与肿瘤靶细胞的培养时的细胞毒性T细胞。此方法采用致密的，弹性体子纳升井（〜100,000井/数组）的数组，在空间上局限在T细胞和靶细胞，在规定的比例，和被耦合到的荧光显微镜来监测效靶共轭和随后的细胞凋亡。

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 ...

quantitative-high-throughput-single-cell-cytotoxicity-assay-for-t

Research

JoVE Journal

Immunology and Infection

14.9K 次观看.  University of Houston. 我们描述了一种单细胞的高通量的检测，以测量与肿瘤靶细胞的培养时的细胞毒性T细胞。此方法采用致密的，弹性体子纳升井（〜100,000井/数组）的数组，在空间上局限在T细胞和靶细胞，在规定的比例，和被耦合到的荧光显微镜来监测效靶共轭和随后的细胞凋亡。

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

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

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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 Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. 
Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High throughput assays are presented that in combination provide excellent tools to quantitate NET release from human neutrophils.

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Methods Article

定量高通量单细胞的细胞毒性T细胞的测定