Name: quantitative high-throughput single-cell cytotoxicity assay for t cells
Uploaded: 2013-02-02T14:00:00
Description: Watch this Scientific Journal Video about Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells at JoVE.com

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...

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	<li>Louis, C. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+and+long-term+fate+of+chimeric+antigen+receptor-positive+T+cells+in+patients+with+neuroblastoma.">Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.</a> Blood. 118, 6050-6056 (2011).</li><li>Savoldo, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+costimulation+improves+expansion+and+persistence+of+chimeric+antigen+receptor-modified+T+cells+in+lymphoma+patients.">CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients.</a> J. Clin. 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Protoc. 1, 1507-1516 (2006).</li><li>Varadarajan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+single-cell+analysis+of+human+CD8++T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Ogunniyi, A. O., Story, C. M., Papa, E., Guillen, E., Love, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+individual+hybridomas+by+microengraving+to+discover+monoclonal+antibodies.">Screening individual hybridomas by microengraving to discover monoclonal antibodies.</a> Nat. Protoc. 4, 767-782 (2009).</li><li>Streeck, H., Frahm, N., Walker, B. 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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 ...

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