这项工作是由来自美国国立卫生DK074656和AI56374（CEM）研究院的资助，以及青少年糖尿病研究基金会的支持。

1。细胞的制备<ol><li>靶细胞培养：鼠标β细胞NIT - 1和NIT - 4的文化，是先前描述的1,2，补充10％胎牛血清，0.02％BSA，非必需氨基酸，15MM的HEPES，16.5毫米的DMEM Glocose和青霉素/链霉素。 51铬标签，种子细胞在平底96以及在5X10 4细胞培养板/ 200μL培养液中。对于显微镜的实验中，8以及实验室德二腔玻璃罩的种子细胞在5X10每室 4 500μL培养基，并允许增长前48小时使用的细胞毒性实验。 </li><li>控制T细胞株培养：为阴性对照使用的Jurkat细胞株（ATCC，CD3 +人体淋巴细胞的细胞系） 。 10％胎牛血清的RPMI1640培养Jurkat细胞，2毫米L -谷氨酰胺，1.5 g / L的碳酸氢钠，10毫米的HEPES和1.0 mm丙酮酸钠。 </li></ol> 2。收集和效应自身反应性CD8 + T细胞的活化 NOD.Cg RAG1 tm1Mom TG（TcraAI4）1Dvs/DvsJ [NOD AI4a NOD.Cg RAG1 tm1Mom TG（TcrbAI4）1Dvs/DvsJ的NOD AI4b]杰克逊实验室（ME）的巴尔港，均购自孕育在我们的鼠标设施。从对NOD AI4a交配F1杂交后代的NOD - AI4b [NOD - AI4a / B]发展在3-5周龄糖尿病。所有小鼠饲养在无特定病原体设施和有关机构的动物护理和使用委员会批准。 <ol><li>安乐死3-4周龄的NOD - AI4a / B小鼠使用的CO 2。 </li><li>收集从小鼠脾脏。安乐死的老鼠准备用乙醇喷雾和开放手术引擎盖下。脾脏被删除，并立即在冰冷的HBSS缓冲。 </li><li>收集脾细胞，用7 mL玻璃匀浆。两到三个脾脏每5毫升冷的HBSS，并与7 mL的玻璃匀浆器匀浆。匀浆是收集和离心5分钟，在1200RPM 4℃用一个Sorvall RT7 +离心机。收集细胞沉淀。 </li><li>删除红血细胞，用低渗溶液处理。颗粒5毫升低渗冰5分钟的解决方案/脾治疗，然后加入同等体积的HBSS中瓦解。离心收集细胞，就像上面，并在50毫升的HBSS悬浮。穿过细胞过滤器悬浮细胞，计数TrypanBlue染色使用一个血球。 </li><li>激活细胞。使用3天的文化与0.1微米AI4模拟表位（氨基酸序列YFIENYLEL）和25 U / mL的IL - 2的RPMI1640悬浮在5X10 6 / mL和激活细胞补充含10％胎牛血清，2毫米L -谷氨酰胺，1.5克/ L碳酸氢钠，10毫米的HEPES和1.0 mm丙酮酸钠。 </li></ol> （3）51铬释放法研究慢性粒细胞白血病<ol><li>标签靶细胞：新增51班的目标细胞在1μCi/孔和文化3个小时，并用培养液洗3次。 </li><li>暂停效应细胞在RPMI 1640培养基，在步骤2.5中提到，使最终体积，每孔加入200μL。 </li><li>最后一次洗涤后标记的靶细胞，添加活化的效应细胞的目标，在预期的效应细胞培养目标（E：T）比值。我们通常用E：T比例为0.4:1，1:1，2:1，5:1，10:1，20:1和50:1。 </li><li>添加新鲜修改RPMI 1640培养基（见第2.5步）6井的靶细胞，作为一种自发的裂解控制。 </li><li>效应和靶细胞共培养16小时。 </li><li>在6x50毫米石灰玻璃管，收集上清</li><li>加入100微升2％的SDS和几次轻轻吹打裂解细胞。 </li><li>另一套6x50毫米石灰玻璃管收集细胞裂解液。 </li><li>洗井一次，用干净的H 2 O，并添加洗细胞裂解液管。 </li><li>上清和细胞裂解液，使用伽玛计数器计数。 </li><li>具体裂解计算如下： <img src="files/ftp_upload/2724/2724eq1.jpg" alt="公式1" /></li></ol> 4。细胞染色活细胞成像其中最常用的线粒体膜电位的染料，TMRM展品至少线粒体毒性。因此，我们选择TMRM这些活细胞成像研究。 <ol><li>准备在DMSO和储存在-20 ° C 40毫米股票TMRM解决方案请[25 NM]酚红的DMEM的解决方案，所有的NIT - 1细胞培养1补充新鲜的工作。 </li><li>目标NIT - 1细胞TMRM 25纳米30分钟染色在37 ° C。 </li><li>替换含有5 nm的TMRM保持均衡分布的染料4新媒体的媒体。 </li><li>共激活T细胞效应AI4使用hematocytometer。 </li><li>染色与1μL/毫升Picogreen为激活效应T细胞AI45分钟，在室温下洗无酚红培养基的3倍。 </li></ol> 5。评估T淋巴细胞介导的​​β细胞死亡，使用活细胞成像<ol><li>山染的NIT细胞阶段的Zeiss LSM 510共聚焦显微镜腔玻璃罩。删除一个dremmel和气体消毒etheline氧化腔玻璃标签。舞台配备了一个活细胞成像室温度控制在37 ° C和5％的CO 2与水分。 </li><li>新增激活和染色的T细胞在不同的E指定钱伯斯：T比率。 </li><li>新增染Jurkat细胞相同数量的控制室内。 </li></ol> 6。获取活细胞图像该显微镜是配备机动阶段，让我们在不同的地点，在实验过程中，自动从相同或不​​同的商会多次获得图像。 <ol><li>使用63X油物镜，在共300帧，每个位置，每3分钟的间隔图像。 </li><li>检测TMRM染色使用的排放量565-619 nm的激发波长543纳米的长度和过滤器设置</li><li>检测Picogreen染色使用的激发为488 nm和过滤器设置为排放量500-630纳米。使用蔡司LSM的软件创建的视频。 </li></ol> 7。出版的视频转换<ol><li>使用蔡司LSM的软件，创建未压缩的AVI格式的电影文件。 </li><li>要发布的格式转换成视频，视频导入到软件包斐济（ <a href="http://pacific.mpi-cbg.de/wiki/index.php/Fiji">http://pacific.mpi-cbg.de/wiki/index.php/Fiji</a> ），ImageJ（ <a href="http://rsb.info.nih.gov/ij/">http://rsb.info。 nih.gov / IJ /</a> ）分配，使用基因位点（Bioformats插件<a href="http://www.loci.wisc.edu/software/bio-formats">http://www.loci.wisc.edu/software/bio-formats</a> ）。 </li><li>调整亮度和对比度设置，以平衡的绿色和红色信号和帧速率设置为2帧/秒，使用“动画选项”菜单。 </li><li>作物的视频512 x 512像素和出口为未压缩的AVI文件。 </li><li>打开这个AVI文件在软件的QuickTime 7（苹果电脑，苹果CA），并在打开的视频文件格式H264/MPEG-4的压缩和出口在1200 KBS /秒。 </li></ol> 8。代表性的成果： <ol><li>一个代表51班慢性粒细胞白血病检测结果。图1显示了代表％具体裂解（Y轴）的效应增加了慢性粒细胞白血病检测结果：目标比例（X轴）增加。靶细胞免疫缺陷的NOD孤立的小岛小学RAG1 - / -小鼠和标有 51班。 NOD.AI4α/β的转基因小鼠的脾细胞的分离和激活在上述步骤2中所述。目标和效应，共同培养16小时。具体裂解百分比计算，在3.11以上步骤中所述。 </li><li>两个有代表性的T细胞介导的​​β细胞线粒体膜电位耗散的结果（正面和负面的）。细胞染色和反应都记录在步骤4，第5和第6段所述，在第7步中创建的视频文件。视频1：小鼠β细胞株NIT - 1染色的线粒体膜电位染料TMRM（红）。激活，自身反应性CD8 + T细胞AI4（Picogreen染绿）共培养这些NIT - 1细胞在E：T比值为50:1。 NIT - 1细胞线粒体膜电位逐渐消退，在400分钟的持续时间，但只在AI4 T细胞相互作用的集群。视频2：作为一个对照实验中，NIT - 1细胞染色：TMRM和Picogreen一个E染色Jurkat细胞共培养T比值为50:1。 Jurkat细胞是人类的T细胞是MHC的不匹配的行。 NIT - 1细胞保持整个线粒体膜电位的整个持续时间400分钟的合作潜伏期。 Jurkat细胞没有交互NIT - 1集群，因此，不诱发杀人。 </li></ol><img src="files/ftp_upload/2724/2724fig1.jpg" alt="图1" /> 图1代表的慢性粒细胞白血病的结果从免疫缺陷NOD.Rag1 /孤立的靶细胞的主要胰岛- NOD.AI4α/β的转基因小鼠的脾细胞和小鼠。目标和效应，共同培养16小时。具体裂解百分比增加的效应：目标比例的增加。 视频1。小鼠β细胞株NIT - 1染色的线粒体膜电位染料TMRM（红）。激活自身反应性CD8 + T细胞AI4（Picogreen染绿）共培养这些NIT - 1细胞在E：T比值为50:1。 NIT - 1线粒体膜电位的耗散泰德逐渐在400分钟的时间，但只有在绿色AI4 T细胞相互作用的集群<a href="http://www.jove.com/files/ftp_upload/2724/video.mov">。点击这里观看</a>视频。 视频2。NIT - 1细胞染色一样在视频1和联合培养Picogreen染人类T细胞株Jurkat描述。 NIT - 1细胞能够保持线粒体膜电位想到了400分钟的时间。 Jurkat细胞没有交互NIT - 1集群的，因此不会诱发<a href="http://www.jove.com/files/ftp_upload/2724/Video2.mp4">杀人，点击这里</a>观看视频。

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动物实验是由佛罗里达体制的​​动物护理和使用委员会大学规定的准则和法规的规定执行。

细胞毒性T细胞介导的β细胞的死亡是主要的病理生理 T1D 5。允许我们使用51铬释放的慢性粒细胞白血病实验研究的效靶反应 6程度。然而，T细胞介导的​​β细胞死亡的详细过程和途径仍然不能完全理解。由于线粒体是β细胞功能和死亡7的关键，我们专注于线粒体的变化，在视觉慢性粒细胞白血病。线粒体膜电位的耗散是一个早在导致细胞凋亡 8的线粒体功...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>铬51 </td><td> Perkin Elmer公司</td><td> NEZ030500IMC </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-60 </td><td></td></tr><tr><td>四甲基若丹明甲酯（TMRM） </td><td> Invitrogen公司</td><td> T668 </td><td></td></tr><tr><td> Picogreen </td><td> Invitrogen公司</td><td> P7581 </td><td></td></tr><tr><td> AI4模拟表位</td><td> mimotopes.com </td><td>氨基酸序列：YFIENYLEL </td><td></td></tr><tr><td> IL - 2 </td><td>研发</td><td> 485英里</td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11885 </td><td></td></tr><tr><td>胎牛血清</td><td> Hyclone公司</td><td> SH30071.03 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A8412 </td><td></td></tr><tr><td> HEPES </td><td> Cellgro </td><td> 25 - 060 - CL </td><td></td></tr><tr><td> MEM非必需氨基酸</td><td> GIBCO公司</td><td> 11140 </td><td></td></tr><tr><td>青霉素/链霉素</td><td>双子座</td><td> 400-109 </td><td></td></tr><tr><td>酚红的DMEM </td><td>西格玛</td><td> D5303 </td><td></td></tr><tr><td> CO2培养箱</td><td>赛默飞世尔</td><td> HeraCell 150i </td><td>保持无菌细胞培养</td></tr><tr><td>生物安全柜</td><td>赛默飞世尔</td><td>备考1440 </td><td></td></tr><tr><td>一次性培养管，6x50毫米石灰玻璃</td><td>费舍尔</td><td> 14 - 958 - A </td><td></td></tr><tr><td>伽玛计数器</td><td> Perkin Elmer公司</td><td>精灵1470自动伽马计数器</td><td></td></tr><tr><td>共聚焦显微镜</td><td>蔡司</td><td> LSM的510元共焦</td><td>机动阶段和活细胞室</td></tr><tr><td>移液器（液1ml，200μL，20μL，10μL，2μL） </td><td> Eppendorf公司</td><td></td><td></td></tr><tr><td>管（黄色） </td><td>费舍尔</td><td> 50819772 </td><td></td></tr><tr><td>离心分离</td><td> Sorvall传奇RT + </td><td> 75004377 </td><td></td></tr><tr><td> Hemacytometer </td><td> Fisher Scientific则</td><td> 267110 </td><td></td></tr><tr><td>直立式显微镜</td><td>蔡司</td><td> Axioskop40 </td><td></td></tr><tr><td> NOD.Cg RAG1 tm1Mom TG（TcraAI4）1Dvs/DvsJ小鼠</td><td>杰克逊实验室</td><td> 004347 </td><td></td></tr><tr><td> NOD.Cg RAG1 tm1Mom TG（TcrbAI4）1Dvs/DvsJ小鼠</td><td>杰克逊实验室</td><td> 004348 </td><td></td></tr><tr><td> NOD -AI4α/βF1小鼠</td><td> N / A </td><td> N / A </td><td>良种在用友动物设施</td></tr></tbody></table>

methods to assess beta cell death mediated by cytotoxic t lymphocytes

1型糖尿病（T1D）是一种T细胞介导的​​自身免疫性疾病。期间发病，患者会逐渐变得更insulinopenic胰岛素生产丢失，想必这胰腺β细胞的杀伤性T细胞的结果。 T1D发展过程的了解β细胞死亡的机制提供见解产生本病的有效的治疗方法。细胞介导的淋巴细胞毒（CML）的检测在历史上所使用的放射性核素铬51（51铬）标记的靶细胞。这些目标都暴露在效应细胞和靶细胞释放51班读淋巴细胞介导的细胞死亡的迹象。抑制剂细胞死亡减少51铬释放的结果。 作为效应细胞，激活自身反应性克隆人口的CD8 +细胞毒性T淋巴细胞（CTL）从鼠标股票转基因为AI4 T细胞受体（TCR）的α和β链隔离。 AI4激活T细胞，51铬标记的目标为16小时的NIT细胞共同培养， 释放 51铬记录来计算具体的裂解在许多重要的生理活动，如能源生产，调节信号转导，细胞凋亡，线粒体参与。 β细胞线粒体功能的改变在T1D发展的研究是一个新的领域的研究。利用线粒体膜电位的染料甲基若丹明甲酯（TMRM）和共聚焦显微镜活细胞成像，我们监测随着时间的推移在β细胞系NIT - 1的线粒体膜电位。对于影像学检查，效应AI4 T细胞标记的荧光核染色染料Picogreen。 NIT - 1细胞和T细胞共培养，在墓室玻璃罩，并安装在一个活细胞室配备显微镜阶段，控制在37 ° C，5％的CO 2和加湿。在这些实验中的图像，每个群集为400分钟，每3分钟。 超过400分钟的过程中，我们观察到的NIT - 1细胞AI4 T细胞连接集群中的线粒体膜电位的耗散。在NIT - 1细胞共培养与MHC的同步控制实验MIS匹配的人体淋巴细胞Jurkat细胞，线粒体膜电位保持完好。这种技术可以用来观察在受到攻击的细胞毒性淋巴细胞，细胞因子或其他细胞毒性试剂的细胞线粒体膜电位的实时变化。

Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

细胞介导的​​淋巴细胞毒（CML）的检测，可用于测试自身反应性的反应，并在体外研究细胞死亡的机制。然而，利用活细胞共聚焦显微成像技术与荧光染料，类型和细胞死亡以及途径利用动力学可以更详细的研究。

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

methods-to-assess-beta-cell-death-mediated-by-cytotoxic-t-lymphocytes

Research

JoVE Journal

Immunology and Infection

13.3K 次观看.  University of Florida. 细胞介导的​​淋巴细胞毒（CML）的检测，可用于测试自身反应性的反应，并在体外研究细胞死亡的机制。然而，利用活细胞共聚焦显微成像技术与荧光染料，类型和细胞死亡以及途径利用动力学可以更详细的研究。

视频: Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

科研

免疫与感染

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

径向移动性和逆转录病毒复制的载体转导，非粘附Alloresponsive T细胞的杀伤功能

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

用末端转移酶介导的 dUTP 尼克末端标记 (Caspase 3/7) 测定青蛙壶菌病的表皮细胞死亡

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...