Name: in situ detection of autoreactive cd4 t cells in brain and heart using major histocompatibility complex class ii dextramers
Uploaded: 2014-08-01T14:00:00.000+00:00
Duration: 13 min 10 s
Description: Watch this Scientific Journal Video about In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers at JoVE.com

这项工作是由美国心脏协会和儿童心肌病基金会（SDG2462390204001）和美国国立卫生研究院（HL114669）的支持。 CM是荣获由心肌炎基金会，新泽西州的博士后研究奖学金补助金的获得者。

伦理声明: 所有动物的程序均按照实验动物的护理和使用的指导原则进行，并经内布拉斯加林肯，内布拉斯加州林肯大学。 1，EAE的诱发<ol><li>以诱导EAE，准备在11所述进行了以下修改的肽/完全弗氏佐剂（CFA）乳化: 步骤1.1:制备PLP 139-151以1毫克/毫升在1×磷酸盐缓冲盐水（PBS）中的浓度步骤1.4:用200微升CFA乳液含有每只动物100微克的PLP 139-151的步骤1.4.1:免疫10的动物，通过混合1毫升PLP 139-151（1毫克/毫升在1x准备2毫升乳液。PBS）和CFA等体积的步骤1.5:两个腹股沟区（75〜每微升）和五到六周龄胸骨区域（约50μl）注入200μL乳液的分割剂量皮下注射FEMA乐SJL / J小鼠。步骤1.7和1.8不适用于诱导EAE的。 </li></ol> EAE小鼠和Cerebrums的收获2。临床评分<ol><li>监测动物外观的临床体征，并且每天从第9天免疫后的得分与疾病的严重程度如下:0 - 健康; 1 - 柔弱的尾部或后肢无力，但不能同时使用; 2 - 柔弱的尾部和后肢无力; 3 - 后肢局部麻痹; 4 - 后肢完全瘫痪; 5 -濒死或死亡12。 </li><li>安乐死的小鼠，当它们达到使用CO 2中的神经学评分为3或4。安乐死的小鼠，先用CO 2的预填充安乐死室转动的CO 2罐不超过6磅的调节，并允许动物留在室内约5分钟，以达到彻底窒息。浸泡在实施安乐死的动物中70％的酒精。立即，将动物上的吸收垫，并用对消毒为牛肝菌和剪刀通过胸腔切割暴露的心。穿刺右耳廓和注入10毫升冷的1X PBS进入心中的左心室灌注。 </li><li>挖掘出的头骨，利用一对弯曲的剪刀剪断的头盖骨以循环方式，并小心地，翻转骷髅骨头用钳子。受钝性分离，并用干净的手术刀，从单独的小脑大脑收获的大脑。将大脑中含有冷的1×PBS管和离开管置于冰上，直到处理。 </li></ol> 3， 在脑部分与MHC II类（IA S）/ PLP 139-151 Dextramers的原位染色<ol><li>制备4％的PBS缓冲低熔点琼脂糖，熔融，加热至50℃并保持在40℃的水浴中，直到使用。 </li><li>将大脑中的一次性深基模组织学卡带保持在冰上，浇上熔融（40℃）的琼脂糖。随即，按大脑轻轻用钳子淹没的组织。将磁带留在冰上15-20分钟，直到凝固完成。 </li><li>用冷冻的1X PBS填充vibratome浴。填充空间周围vibratome浴冰块保持PBS中0和2之间的温度 °C。固定清洁刀片进入vibratome和设置在叶片角度为15°。 </li><li>组织在vibratome位置。修剪多余的琼脂糖从包埋的组织的各个侧面及稍微露出大脑的腹面。 <ol><li>涂片用超级胶水vibratome组织块并通过将大脑的暴露腹面过胶横向切片转移琼脂糖包埋的组织在胶合表面上。别动组织，一旦组织被设置在该块。 </li><li>上放置装有冰的琼脂糖包埋组织的vibratome块，并让胶干燥15分钟。 </li></ol></li><li>在此期间，将组织湛的BER每孔一个24孔板。填装有组织腔用1ml冷的1×PBS的孔中，并离开该板在冰上。 <ol><li>通过将一个薄尼龙网用强力胶25毫升聚丙烯移液管的切断端准备组织商会;修剪多余的网格围绕吸管后，从网格切吸管为1厘米的长度。 </li></ol></li><li>降低vibratome叶片组织的顶表面的水平;设置vibratome速度0.22毫米/秒，并开始向前运动切片，使200微米厚的切片。每转用水彩软刷到组织室放置在24孔板的孔中以及在一侧的大脑部分。保持板置于冰上，直到切片完毕，以防止组织退化。注:请按照以下步骤从大脑背侧向腹侧表面的三个不同区域（ 图2）取得合共三次配对部分。从一个部分放置在三个单独的组织腔室的每对（总共三个部分）被指定为与特定dextramers染色（IA 秒/ PLP 139-151）和抗-CD4。同样地，另一组三个部分被放置在三个单独的组织腔室被指定用于与控制dextramers染色（IA 秒/ TMEV 70-86）和抗-CD4。 </li><li>传送含有指定用于特定dextramers在染色成三个孔在24孔板中与一个腔室，每孔的部分中的3组织室的，300微升含别藻蓝蛋白鸡尾酒（APC）缀合的PLP 139-151 dextramers（2.5微克/毫升）和抗-CD4-藻红蛋白（PE，5微克/毫升）在封闭液（1×PBS中，用2％正常山羊血清）先前的溶液。盖井有盖。 </li><li>同样，含有转指定用于控制dextramers染色分为三个井在24孔板与我们每一个室的部分三个组织商会LL，其中，300微升含APC标记TMEV 70-86 dextramers（2.5微克/毫升）和抗-CD4-PE（5微克/毫升）在封闭液鸡尾酒的以前添加。盖井有盖。注:请参阅Massilamany 等人 ，8准备dextramers，而葡聚糖分子准备dextramers被好心Immudex，哥本哈根，丹麦提供。 </li><li>标签的井，和包裹该板用铝箔，然后将板在摇摆平台设置为平缓旋转1.5小时在黑暗中在RT上。 </li><li>孵育期后，洗涤组织切片在摇动平台上，通过将所述组织腔到新鲜的孔含有1ml冷的1×PBS中，并避免运球1中的内容以及到另一个。重复洗涤三次，让每冲洗至少10分钟。确保组织切片没有得到干燥的，因为它们会粘在腔室的侧面。 </li><li>通过将组织湛修复部分的BER到一个新的井在24孔板中含有2小时，轻轻旋转摇摆平台上过滤4％的PBS缓冲的多聚甲醛（pH 7.4）中1毫升。 </li><li>如步骤3.10所述重复洗涤三次。 </li><li>用水彩软毛刷放在一个干净的显微镜载玻片染色和固定部分。擦去多余的水不接触的部分，并安装使用的安装介质中的一节。盖上盖玻片，显微镜，让幻灯片在室温O / N干在黑暗的房间。 </li><li>保存幻灯片在不透光的滑动储物盒，在4°C。 </li></ol> 4，感应EAM心和收集<ol><li>诱导EAM如参考图11所述。 </li><li>安乐死在使用清洁室装有二氧化碳气瓶21天接种后的小鼠，和浸泡的动物在70％的酒精。随即，将动物上的吸收垫，并用钳子和剪刀暴露心脏s由通过胸腔切割。穿刺右耳廓和注入10毫升冷的1X PBS进入心中的左心室灌注。收集的心和地方在含冷的1X PBS管。 </li></ol> 5， 在心脏部分与MHC II类（IA K）/ MYHC 334-352 Dextramers的原位染色<ol><li>制备4％的PBS缓冲低熔点琼脂糖，通过加热熔化，在50℃，并让它在40 &#160; °C的水中洗澡，直到使用。 </li><li>将心脏在一次性深基模组织学卡带放在冰上，倒入熔化的40℃的琼脂糖直至在步骤3.2中所述的组织被完全淹没。 </li><li>按照步骤3.3。 </li><li>修剪多余的琼脂糖从包埋组织的所有侧面和微微露出心脏的基础。用涂抹强力胶vibratome组织块和放置的暴露表面转移琼脂糖包埋组织在表面粘心脏的胶水。别动组织，一旦组织被设置在该块。 <ol><li>上放置装有冰的琼脂糖包埋组织的vibratome块，并让胶干燥15分钟。 </li></ol></li><li>按照步骤3.5和3.6。 </li><li>用水彩软刷到组织室置于24孔板的孔中转移的心脏部分。保持板置于冰上，直到切片完毕，以防止组织退化。注:请按照以下步骤获得合共八对部分从顶点到心脏基地（ 图2）。将组织商会的所有部分，分配高达每室3部分。从每对（共8部分）的一个部分被指定用于特定dextramers（IA K / MYHC 334-352）和抗-CD4染色。同样地，另一组八个部分被指定用于与控制dextramers（IA K / RNA酶43-56）及抗CD4染色。 </li><li>转让包含指定用于特定dextramers染色成三个孔在24孔板中，每孔1室中的部分3的组织腔的，300微升含APC标记MYHC 334-352 dextramers（2.5微克/毫升）的鸡尾酒和抗-CD4-PE（5微克/毫升）在封闭液中预先加入。盖井有盖。 <ol><li>同样，传输包含指定用于控制dextramers染色成三个孔在24孔板中以每孔1室中的部分中的3组织腔，其中，300微升含APC标记的RNase 43-56 dextramers（2.5微克鸡尾酒/ ml）和抗-CD4-PE（5微克/毫升）在封闭液中预先加入。盖井有盖。 </li></ol></li><li>标签的井，和包裹该板用铝箔，然后将板在摇摆平台设置为平缓旋转1.5小时在黑暗中在RT上。 </li><li>按照步骤，3.10至3.11 </li><li>重复洗涤THR如步骤3.10中描述的冰。 </li><li>将染色和固定部分（最多三段/幻灯片）上用水彩软毛刷清洁显微镜幻灯片。擦去多余的水不接触的部分，并使用安装介质安装部分。盖上盖玻片，显微镜，让幻灯片在室温O / N干在黑暗的房间。 </li><li>保存幻灯片在不透光的滑动储物盒在4 °C。 </li></ol> 6。Dextramer +分析（DEXT +）CD4 + T细胞通过激光扫描共聚焦显微镜（LSCM）（以双方共同的大脑和心脏部分） <ol><li>使用尼康A1-Eclipse的90I共聚焦显微镜系统进行例行扫描和采集图像。要启动单击打开，NIS元素的AR软件（4.13版）。 <ol><li>选择"TRITC"，并从面板"Cy5的"通道。分别为TRITC和Cy5通道的激发/发射波长，561.5纳米/ 553-618 nm和640.7纳米/ 663-738 nm的默认显示。 </li><li>为TRITC（CD4-PE）和Cy5（dextramer-APC）渠道分配pseudocolors"绿色"和"红色"，分别。 </li><li>将要检查在显微镜舞台上的幻灯片和下放大100倍手动对焦。设置"HV"110和偏移为"0"。点击"焦点访谈"和调整"电压"来优化激光器。 </li><li>点击"焦点访谈";扫描截面从顶部到底部，并设置"Z"电平，用于图像采集。点击"CH系列"和获取图像的顺序。识别DEXT + CD4 + T细胞中出现的基础上共同定位的同时从红色（dextramers）和绿色（CD4 +）通道产生的信号，为黄色斑点状细胞。 </li></ol></li><li>使用奥林巴斯FV500-BX60共聚焦显微镜DEXT + CD4 + T细胞的易定量枚举。开始单击打开，FLUOVIEW软件（版本4.3）。 <ol><li>从下拉菜单中选择染料"Cy3标记"和"Cy5标记"，然后单击"应用"来加载相应的激光器。注意:在激发/发射波长为Cy3标记（543纳米/伪彩色的"绿色"，CD4-PE）和Cy5（633纳米/伪彩色的"红色"; dextramer-APC）的信道。 </li><li>将要检查在显微镜幻灯片的幻灯片，并在低倍率（4X或10X）手动对焦了。点击"焦点访谈"，而Cy3的激光，并专注于炎症病灶。 </li><li>改变客观上放大100倍。从下拉菜单中设置文件大小为"五百一十二分之五百十二"。点击"焦点访谈"，调整为"PMT"，"增益"的价值观和"偏移"参数分别优化激光器Cy3和Cy5。 </li><li>点击"Z级"，然后单击"焦点访谈"而无论是激光器上。集在"Z载物台"的通过点击"向上"和"向下"箭头的厚度。将"Z"的时间间隔为2微米。 </li><li>点击"停止"，并选择"Seq123"。然后，单击"XYZ"，并开始采集"Z"系列图像内的炎症病灶选定的区域。拯救"Z序列图像"为AVI文件。要保存图像文件，从"Z系列图像"选择图像，然后另存为"TIFF"格式。 </li></ol></li><li>检查从其中一组三段染了IA 秒/ PLP 139-151 dextramers/anti-CD4，另一组三个部分与执行机构的S / TMEV 70-86 dextramers/anti-CD4执行大脑3成对的节定量分析。 <ol><li>同样，分析对心脏八对的部分，其中，一组八个部分沾上了IA K / MYHC 334-352 dextramers/anti-CD4和另一个SE吨八个部分用RNase 43-56 dextramers/anti-CD4。 </li></ol></li><li>每检查大脑中第10至15炎性病灶，并在每个重点，掌握了一套15〜25序列图像顺序。 <ol><li>检查共20炎性病灶从所有八个部分心脏，并从每个重点掌握10〜15序列图像顺序。通过扫描载玻片在一个"水平 - 垂直向上，水平 - 垂直向下"运动，以便避免在同一焦点的重复的"Z"系列走&#39;Z&#39;的序列图像。 </li><li>一旦"Z"连续拍摄多张照片，通过识别幻灯片编号，各部分的名称，以及所采取的"Z"系列的图像数命名文件。 <ol><li>使用&#39;ImageJ的"软件计算DEXT + CD4 + T细胞相对于CD4 + T细胞的总数量。选择"ImageJ的的&#39;类型的计数器"，检查＆＃8216;显示所有"，并开始通过点击每个小区的中心算起，继续在不同的计数"使用一侧的箭头，Z&#39;的序列图像。计算所有的CD4 + T细胞后，选择其他类型的计数器和计数的CD4 + DEXT + T细胞。单击结果选项卡以获取细胞与两个不同的计数器计数的总数。 </li><li>取得的总数，增加细胞中的所有"Z"序列表示所有三个脑的部分或所有的8心脏切片的图像的数目。 </li></ol></li></ol></li></ol>

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(88), e51654 (2014).</li><li>Massilamany, C., Steffen, D., Reddy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epitope+from+Acanthamoeba+castellanii+that+cross-react+with+proteolipid+protein+139-151-reactive+T+cells+induces+autoimmune+encephalomyelitis+in+SJL+mice.">An epitope from Acanthamoeba castellanii that cross-react with proteolipid protein 139-151-reactive T cells induces autoimmune encephalomyelitis in SJL mice.</a> J Neuroimmunol. 219 (1-2), 17-24 (2010).</li><li>Donermeyer, D. L., Beisel, K. W., Allen, P. M., Smith, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocarditis-inducing+epitope+of+myosin+binds+constitutively+and+stably+to+I-Ak+on+antigen-presenting+cells+in+the+heart.">Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.</a> J Exp Med. 182 (5), 1291-1300 (1995).</li><li>Liao, L., Sindhwani, R., Leinwand, L., Diamond, B., Factor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+alpha-myosin+heavy+chains+differ+in+their+induction+of+myocarditis.+Identification+of+pathogenic+epitopes.">Cardiac alpha-myosin heavy chains differ in their induction of myocarditis. Identification of pathogenic epitopes.</a> J Clin Invest. 92 (6), 2877-2882 (1993).</li><li>Cameron, T. O., Cochran, J. R., Yassine-Diab, B., Sekaly, R. P., Stern, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+detection+of+antigen-specific+CD4++T+cells+by+HLA-DR1+oligomers+is+dependent+on+the+T+cell+activation+state.">Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state.</a> J Immunol. 166 (2), 741-745 (2001).</li><li>Novak, E. J., 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=Activated+human+epitope-specific+T+cells+identified+by+class+II+tetramers+reside+within+a+CD4high,+proliferating+subset.">Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset.</a> Int Immunol. 13 (6), 799-806 (2001).</li><li>Reddy, J., 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=Detection+of+autoreactive+myelin+proteolipid+protein+139-151-specific+T+cells+by+using+MHC+II+(IAs)+tetramers.">Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers.</a> J Immunol. 170 (2), 870-877 (2003).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> J Immunol. 165 (2), 613-617 (2000).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+tetramer+staining.">In situ tetramer staining.</a> J Immunol Methods. 268 (1), 29-34 (2002).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+staining+using+MHC+class+I+tetramers.">In situ staining using MHC class I tetramers.</a> Curr Protoc Immunol. Chapter 17, Unit 17.14.11 (2004).</li></ol>

没有利益冲突被宣布。

与MHC I类四聚体，采用MHCⅡ类四聚体的常规应用仍然是具有挑战性的，尤其是枚举低亲和力的自身反应性CD4 + T细胞，部分原因是由于其激活依赖6,15-17的前体频率。最近，这个问题是由创建下一代四聚体，称为规避"dextramers，"允许我们检测抗原特异性CD4 + T细胞为一些自身抗原，如PLP 139-151，MOG 35-55和MYHC 334 - 352 8。在此报告中，用于第一时间的推移，MHC II类dextramers的实用程序来检?...

使用的主要组织相容性复合体（MHC）Ⅱ类四聚体检测抗原特异性CD4 + T细胞一直是个挑战1-7。为了增强MHC II类四聚体的检测灵敏度，研究小组已经创造下一代四聚体，命名为"dextramers"8。 Dextramers含有葡聚糖主链，其中多个链亲和素-荧光基团缀合，使得一些肽-拴系的，生物素化的MHC单体可以连接到一个葡聚糖分子9。因此，包含数个MHC-肽复合物的MHC dextramers的大的聚集体可以与多个T细胞受体（TCR）进行交互。 这组最近产生dextramers各种自身抗原，并证明dextramers的检测灵敏度约为5倍以上的四聚体8。实验系统包括实验性自身免疫性脑脊髓炎（EAE）诱导蛋白脂质蛋白（PLP）在SJL小鼠139-151; EAE诱导的髓磷脂少突胶质细胞糖蛋白（MOG）在C57BL / 6小鼠35〜55;和实验性自身免疫性心肌炎（EAM）与诱导心肌肌球蛋白重链α（MYHC）中的A / J小鼠334-352段。这项研究表明，使用PLP 139-151的-和MYHC 334-352 dextramers来检测抗原特异性CD4 + T细胞在原位特异性通过开发一个直接染色方案，从而省去了使用荧光抗体放大信号，一接近常用配合使用四聚体。此外，评估组织的综合方法也被设计成可靠地列举抗原特异性CD4 + T细胞在原位 10的频率。检测抗原特异性T细胞在原位的允许由从所造成的旁观者激活特定抗原刺激事件的描述。同样地， 在原位技术也providES机会来跟踪在靶器官的抗原特异性T细胞浸润的动力学。

<table><tbody><tr><td>CFA</td><td>Sigma Aldrich, St Louis, MO</td><td>5881</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>MTB&amp;nbsp; H37Rv extract&amp;nbsp;</td><td>Difco Laboratories, Detroit, MI</td><td>231141</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>PT</td><td>List Biologicals Laboratories, Campbell, CA</td><td>181</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>1x PBS&amp;nbsp;</td><td>Corning, Manassas, VA</td><td>21-040-CV</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Female A/J mice&amp;nbsp;</td><td>Jackson Laboratories, Bar Harbor, ME</td><td>646</td><td></td></tr><tr><td>Female SJL/J mice</td><td>Jackson Laboratories, Bar Harbor, ME</td><td>686</td><td></td></tr><tr><td>Leur-lok sterile 1 ml syrringe</td><td>BD, Franklin Lakes, NJ</td><td>309628</td><td></td></tr><tr><td>Leur-lok sterile 3 ml syrringe</td><td>BD, Franklin Lakes, NJ</td><td>309657</td><td></td></tr><tr><td>Sterile needle, 18 G</td><td>BD, Franklin Lakes, NJ</td><td>305195</td><td></td></tr><tr><td>Sterile needle, 27 1/2 G</td><td>BD, Franklin Lakes, NJ</td><td>305109</td><td></td></tr><tr><td>3-way stopcock&amp;nbsp;</td><td>Smiths Medical ASD, Inc. Dublin, OH</td><td>MX5311L</td><td></td></tr><tr><td>Kerlix gauze bandage rolls&amp;nbsp;</td><td>Covidien, Mansfield, MA</td><td>6720</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional, Roswell, GA</td><td>34155</td><td></td></tr><tr><td>Agarose, Low Melting Point, Analytical Grade</td><td>Promega corporation, Madison, WI</td><td>V2111</td><td></td></tr><tr><td>Immunopure normal goat serum</td><td>Thermo Scientific, Waltham, MA</td><td>31873</td><td>Store at -20 &amp;#176;C</td></tr><tr><td>Anti-mouse CD4 conjugated with PE dye</td><td>eBioscience, San Diego, CA&amp;nbsp;</td><td>12004185</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences, Hatfield, PA</td><td>19202</td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Faramount Aqueous Mounting Medium</td><td>Dako, Carpinteria, CA</td><td>10073021</td><td></td></tr><tr><td>Conical tube (15 ml)</td><td>BD Biosciences, San Jose, CA</td><td>352099</td><td></td></tr><tr><td>Conical tube (50 ml)</td><td>BD Biosciences, San Jose, CA</td><td>352070</td><td></td></tr><tr><td>24-well flat bottomed tissue culture plates</td><td>BD Biosciences, San Jose, CA</td><td>356775</td><td></td></tr><tr><td>Water color #2 brushes</td><td>Charles Leonard, Inc. Hauppauge, NY</td><td>73502</td><td></td></tr><tr><td>Plain Microscope slides, size: 3&quot; x 1&quot; x 1.2 mm</td><td>Fisher Scientific, Pittsburgh, PA</td><td>1255010</td><td></td></tr><tr><td>Premium Cover glass, 22 x 22-1</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12548B</td><td></td></tr><tr><td>Disposable deep base mold histology cassettes</td><td>Laboratory prodcust, Rochester, NY</td><td>M-475-10</td><td></td></tr><tr><td>Loctite Super glue</td><td>Henkel Corporation, Westlake, OH</td><td>1471879</td><td></td></tr><tr><td>Razor blades</td><td>Gillette SuperSilver</td><td></td><td></td></tr><tr><td>Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)</td><td>Neopeptide, Cambridge, MA</td><td></td><td>Store at 4 &amp;#176;C</td></tr><tr><td>PLP 139-151 (HSLGKWLGHPDKF)</td><td>Neopeptide, Cambridge, MA</td><td></td><td>Store at 4 &amp;#176;C</td></tr><tr><td>MHC class II/IA&lt;sup&gt;s&lt;/sup&gt; PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye</td><td></td><td></td><td>Store at 4 &amp;#176;C</td></tr><tr><td>MHC class II/IA&lt;sup&gt;k&lt;/sup&gt; Myhc 334-352 or&amp;nbsp; or RNase 40-56 dextramers conjugated with APC dye</td><td></td><td></td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Dextran conjugated SA-APC</td><td>Immundex, Copenhagen, Demark</td><td></td><td>Store at 4 &amp;#176;C</td></tr><tr><td>Sterile surgical scissors and forceps</td><td>INOX tool Corporation</td><td></td><td></td></tr><tr><td>Micro oven</td><td>GE Healthcare, Pittsuburgh, PA</td><td></td><td></td></tr><tr><td>Leica VT 1200 Vibratome</td><td>Leica Microsystems, Inc. Buffalo Grove, IL</td><td></td><td></td></tr><tr><td>Olympus BX 60&amp;nbsp; Laser scanning confocal microscope</td><td>Olympus America, Inc. Center Valley, PA</td><td></td><td></td></tr><tr><td>Nikon A1-Eclipse 90i confocal microscope</td><td>Nikon Inc. Melville, NY</td><td></td><td></td></tr></tbody></table>

in situ detection of autoreactive cd4 t cells in brain and heart using major histocompatibility complex class ii dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

在这份报告中， 原位检测抗原特异性，自身反应性CD4 + T细胞通过直接染色与MHC II类dextramers的描述。刚割下的脑切片均来自EAE小鼠并用含PLP 139-151（具体的）或TMEV 70-86（控制）dextramers和抗-CD4的鸡尾酒。顶部面板在图1所示的costained用PLP 139-151 dextramers（红色）和CD4抗体（绿色），其中双阳性细胞呈黄色脑切片的LSCM分析。这样的功能是没有的沾满TMEV 70-86（控制）dextramers（底板）?...

Watch this Scientific Journal Video about In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers at JoVE.com

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

通过直接染色与主要组织相容性复合物Ⅱ类dextramers检测自身反应性CD4 + T细胞在大脑和心脏的协议已经本报告中进行了描述。为综合分析，一种可靠的方法来枚举抗原特异性CD4 + T细胞在原位的频率也被设计出来。

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

in-situ-detection-autoreactive-cd4-t-cells-brain-heart-using-major

Research

JoVE Journal

Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

11.6K 次观看.  University of Nebraska, Lincoln. 通过直接染色与主要组织相容性复合物Ⅱ类dextramers检测自身反应性CD4 + T细胞在大脑和心脏的协议已经本报告中进行了描述。为综合分析，一种可靠的方法来枚举抗原特异性CD4 + T细胞在原位的频率也被设计出来。 

视频: In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

大脑和脊髓的单核细胞的分离，使用Percoll梯度

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

科研

免疫与感染

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

生物学

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

医学

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

细胞内染色和流式细胞仪识别鼠主动脉，肾脏和淋巴结内淋巴细胞亚群的高血压模型

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

心脏外免疫细胞的分离与鉴定

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

小鼠肺中抗原特异性 T 细胞的分离和表征

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

作者聚焦:使用四聚体检测小鼠肺中抗原特异性 T 细胞的优化方案

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...