Name: methods to assess beta cell death mediated by cytotoxic t lymphocytes
Uploaded: 2011-06-16T12:00:00
Description: Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

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...

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	<li>Hamaguchi, K., Gaskins, H. R., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIT-1,+a+pancreatic+beta-cell+line+established+from+a+transgenic+NOD/Lt+mouse.">NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.</a> Diabetes. 40, 842-849 (1991).</li><li>Chen, J., Gusdon, A. M., Piganelli, J., Leiter, E. H., Mathews, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mt-Nd2a+modifies+resistance+against+autoimmune+Type+1+diabetes+in+NOD+mice+at+the+level+of+the+pancreatic+beta+cell.">mt-Nd2a modifies resistance against autoimmune Type 1 diabetes in NOD mice at the level of the pancreatic beta cell.</a> Diabetes. , (2010).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys J. 76, 469-477 (1999).</li><li>Lemasters, J. J., Ramshesh, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+mitochondrial+polarization+and+depolarization+with+cationic+fluorophores.">Imaging of mitochondrial polarization and depolarization with cationic fluorophores.</a> Methods Cell Biol. 80, 283-295 (2007).</li><li>Abel, M., Krokowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+immune-mediated+(type+1)+diabetes+mellitus:+potential+for+immunotherapy.">Pathophysiology of immune-mediated (type 1) diabetes mellitus: potential for immunotherapy.</a> BioDrugs. 15, 291-301 (2001).</li><li>Mathews, C. E., Graser, R. T., Savinov, A., Serreze, D. V., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+resistance+of+ALR/Lt+mouse+beta+cells+to+autoimmune+destruction:+role+for+beta+cell-expressed+resistance+determinants.">Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.</a> Proc Natl Acad Sci U S A. , 235-240 (2001).</li><li>Szabadkai, G., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+mediated+cell+death+in+diabetes.">Mitochondria mediated cell death in diabetes.</a> Apoptosis. 14, 1405-1423 (2009).</li><li>Ly, J. D., Grubb, D. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+membrane+potential+(deltapsi(m))+in+apoptosis;+an+update.">The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.</a> Apoptosis. 8, 115-128 (2003).</li><li>Dudek, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-cells+from+T-cell+receptor+transgenic+NOD8.3+mice+destroy+beta-cells+via+the+perforin+and+Fas+pathways.">Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.</a> Diabetes. 55, 2412-2418 (2006).</li><li>Pakala, S. V., Chivetta, M., Kelly, C. B., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+autoimmune+diabetes+the+transition+from+benign+to+pernicious+insulitis+requires+an+islet+cell+response+to+tumor+necrosis+factor+alpha.">In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor alpha.</a> J Exp Med. 189, 1053-1062 (1999).</li></ol>

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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

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

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

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

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

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 ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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

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

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

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 ...