Name: human lung dendritic cells: spatial distribution and phenotypic identification in endobronchial biopsies using immunohistochemistry and flow cytometry
Uploaded: 2017-01-20T14:00:00.000+00:00
Duration: 11 min 2 s
Description: Watch this Scientific Journal Video about Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry at JoVE

笔者想感谢谁临床资料已经促成了这一研究志愿者。我们也感谢工作人员在公共卫生与临床医学，医学/呼吸内科的科，大学医院，于默奥（Norrlands universitetssjukhus）部的所有临床资料的收集。 这项工作得到了补助金从瑞典研究理事会，瑞典心肺基金会，瑞典战略研究基金会，并卡罗琳斯卡医学院AS-S支持。

注:此研究于默奥，瑞典批准了区域伦理审查委员会。 1.支气管镜从人类受试者取样活检支气管<ol><li>获得所有参与者的知情同意。 </li><li>治疗口服咪达唑仑（4-8毫克）和静脉注射格隆（0.2-04毫克）的支气管镜检查前30分钟科目。应用表面麻醉用喉和支气管利多卡因。让我们用〜3个毫升利多卡因4％的受漱口并申请3毫升舌根并经由一个注射器喉喉。优化8-10剂量利多卡因喷表面麻醉。进行这一步的主题会议。 </li><li>通过塑料喉舌与仰卧位的主题将通过口腔灵活的视频支气管镜。使用特制的喷雾导管来完成通过支气管镜远端气管和支气管的局部麻醉。在这里，使用剂量为约5-10毫升2％利多卡因。 </li><li>取从主隆突6-9支气管粘膜活检和使用有孔镊子（ 图1）的主支气管分裂。继支气管镜检查，并离开医院之前，让主题休息2-4小时，并提供他/她的便餐。 </li></ol> 2.在嵌入乙二醇丙烯酸甲酯（GMA）树脂活检注:GMA染色协议在南安普敦大学，组织化学研究组17最初被开发。 <ol><li>固定:对于免疫组织化学，除去从钳子的活检和直接将它们放入装有3毫升冰冷脱水丙酮和蛋白酶抑制剂的玻璃小瓶（苯甲基磺酰氟（35毫克/ 100毫升丙酮）和碘乙酰胺（370毫克/ 100毫升丙酮））。过夜修复组织在-20℃（ 图2）。 注:以下步骤应PERFORmed指在适当的丁腈手套在通风橱。嵌入试剂盒包含可引起过敏，刺激，和/或皮肤过敏反应的组件。所有垃圾必须进行处置危险废物。 </li><li>脱水:用抑制剂取出丙酮并用新鲜的无水丙酮替代（没有抑制剂）在室温（RT）下15分钟。除去丙酮并用甲基苯甲酸代替它15分钟。 </li><li>渗透:准备在乙二醇甲基丙烯酸酯单体5％苯甲酸甲酯溶液处理解决方案; 10毫升足够用于一个小瓶。使用塑料巴斯德移液管，吸出苯甲酸甲酯溶液并用3毫升的处理溶液替换。孵育活检，并在4℃下2小时过量的处理溶液;巴斯德移液器可用于所有后续溶液的改变，包括嵌入溶液。 </li><li>交换处理液每2小时（3×2小时的孵育），所以生物的总浸润时间在该处理溶液psies为至少6小时。插入纸标签进入嵌入胶囊的顶端识别活检。 </li><li>制备的75毫克过氧化苯甲酰在10ml乙二醇甲基丙烯酸酯单体的一个嵌入溶液。加入0.25g毫升N，N-二甲基苯胺的聚（环氧乙烷）的（加速器）以嵌入溶液，开始聚合反应。除去处理溶液和一种活检放入使用镊子的有关嵌入的胶囊。 </li><li>慢慢地填补了嵌入胶囊顶端嵌入与解决方案，并盖上。避免干扰活检和创造大量气泡。在4℃孵育胶囊直至树脂完全聚合。 </li><li>储存在-20℃的嵌入活检中含有约5g硅胶的50毫升管中。 </li></ol> 3. GMA嵌入支气管活检切片<ol><li>显微镜载玻片涂层:洗净显微镜载玻片在洗碗机在一个正常的周期。盖上玻片并风干。 </li><li>制备在蒸馏水中的10％聚-L-赖氨酸（PLL）溶液包衣溶液。淹没在涂液的幻灯片5分钟，然后让他们自然风干。存储在他们的原盒干PLL涂层的幻灯片。 </li><li>在蒸馏水中洗片玻璃带用0.1％吐温20。冲洗，用70％的乙醇和用纸巾擦干玻璃。使用刀制造玻璃条形切割玻璃刀片。存储刀片时，防止移动，以确保切削刃不会被损坏或有缺口的容器。 </li><li>活检微调:将胶囊分配器活检囊采用碳钢单刃刀片每边砍伐。取出GMA包埋活检并牢牢将其放置在副。各地使用钢刀活检修剪多余GMA。 </li><li> GMA切片。 <ol><li>制备用蒸馏水0.05％的氨水溶液。在广场的第玻璃刀和活检Ë切片。仔细使刀片靠在块的表面平坦调整刀架和所述活检块的角度对准与叶片活检块。 </li><li>切割用切片机2微米厚的部分在一个缓慢的切削速度和使用镊子转移和上浮部分的氨水。浮动部分为45-90秒，让温柔的抗原修复和部分展开。 </li><li>拿起显微镜载玻片部分。用甲苯胺蓝染色快速检查活检组织学检查。 </li><li>干燥的热板上集合的幻灯片到50℃，并应用500微升过滤甲苯胺蓝染色，以使用塑料巴斯德吸管的部分。暖节上热板，直到绿环开始在污渍的边缘出现，然后用清水洗净。 </li><li>使用光学显微镜，检查活检组织学检查。用于免疫组化部分应包含完好椎板好的地区和固有上皮细胞，用尽可能少的腺体和小平滑肌越好。 </li><li>正如在3.4.2节，切具有良好的组织切片，并挑选他们与PLL涂层显微镜载玻片的免疫组化染色。从每个切片进行分析切至少两个部分。干片至少1小时。干燥后，部分可以被包裹在锡箔，并储存在-20℃下长达2周，或它们可以立即进行免疫染色。 </li></ol></li></ol> 4. GMA嵌入支气管活检免疫组化染色注:叠氮化钠是有毒的。通风橱内制备0.1％叠氮化钠溶液和从酸路程。与酸接触释放出有毒气体。 <ol><li>周围画用金刚石尖笔段和安排幻灯片到染色架。 </li><li>执行过氧化物块。制备在0.1％叠氮化钠溶液中的0.3％的过氧化氢的过氧化物酶块溶液。申请Y 1毫升过氧化物块部分，并且孵育30分钟。 </li><li>制备0.05M的Tris缓冲盐水（TBS），pH值7.6的洗涤溶液中。洗净的幻灯片在TBS，3 5分钟。 </li><li>准备在DMEM一个的1％BSA封闭液。提前大批量，等分做到这一点，它冻结直到需要。漏的幻灯片，并培育在块溶液区段30分钟以封闭非特异性抗体结合。如果非特异性结合仍然出现，包括来自相同物种作为第二抗体5％血清块30分钟温育步骤。 </li><li>稀释在TBS中的第一抗体（CD45或的CD1a）至所需浓度（CD45在1稀释:1000;的CD1a稀释为1:1.00），并把它应用到幻灯片。盖玻片的章节，并在室温下孵育过夜。 </li><li>在TBS中洗涤载玻片三次。稀释生物素化的次级抗体（针对第一抗体宿主物种，在这种情况下，2）的兔抗小鼠的F（ab&#39;）在TBS至所需浓度（1:300稀释）并将其应用到幻灯片。在室温下孵育2小时。 </li><li>使用前制备链霉抗生物素 - 过氧化物酶复合物至少30分钟。确保有足够覆盖所有幻灯片。洗载玻片在TBS三次。应用溶液的幻灯片和在室温下孵育2小时。 </li><li>洗载玻片在TBS三次。制备3-氨基-9-乙基咔唑（AEC）过氧化物酶底物溶液（每制造商的说明制备）。将其应用到幻灯片和孵育20-30分钟或直到所需的染色强度发展。 </li><li>冲洗载玻片在流动的自来水进行5分钟。染液的部分用2分钟过滤Mayer的苏木解决方案。在流动的自来水进行5分钟洗涤载玻片。 </li><li>沥干幻灯片和覆盖永久安装水介质中的部分。干燥在80℃下将载玻片在干燥炉中。允许的幻灯片冷却，然后使用DPX安装它们并放置盖玻片。 </li></ol> 5.圣癌宁分析<ol><li>使用连接到计算机的安装照相机的光学显微镜在40X放大率分析部分。 注:对于细胞分析，计数阳性染色，核细胞支气管固有层和完整上皮内，不含平滑肌，腺体，大血管，和不匹配的或受损的组织的区域。 </li><li>平均的细胞计数和纠正的平均细胞数为固有层的面积和上皮的长度。固有层和上皮的长度的面积可以使用图像分析程序来计算。 </li></ol> 6.支气管活检酶消化<ol><li>洗涤活检:在含有10ml Hank氏缓冲盐溶液（HBSS）（ 图3）的15毫升锥形管中使用无菌镊子，地点完整活检。以30rpm在室温下孵育5分钟，在摇摆平台上。转移活检到无菌ð通过滗析15ml试管用HBSS的内容杂交。 </li><li>扰乱粘液用DTT:在管用10ml的HBSS含5mM 1,4-二硫苏糖醇（DTT）更换的HBSS。转移活检回用无菌镊子15毫升管。以30rpm在室温下孵育15分钟在摇摆平台上。 </li><li>轻轻涡管15秒。通过滗析15毫升管与HBSS和DTT的内容转移到活检无菌培养皿。 </li><li>传送活检到培养基中:用10ml RPMI 1640培养基的更换HBSS和DTT在管。转移活检回用无菌镊子15毫升管。以30rpm在室温下孵育10分钟在摇摆平台上。 </li><li>消化胶原酶和DNA酶活检。 <ol><li>制备在预热的RPMI含有1M HEPES溶液胶原酶II（0.25毫克/毫升）和DNA酶（0.2毫克/毫升）的消化溶液。加入500μL的每孔消解液在48孔邪教组织URE板。 </li><li>将每孔1活检消化解决方案。孵育60分钟的摇动平台上的板在37℃在220rpm。移液器上下30分钟后，以再分散的活检，并在60分钟后，以完全分解的组织。 </li></ol></li><li>准备单细胞悬液。 <ol><li>池一起消化活组织检查从孔到50毫升管中使其通过一个40微米的细胞滤网。通过用冰冷的FACS缓冲液（磷酸盐缓冲的盐水（PBS）含有2％胎牛血清）洗涤各孔收集剩余的细胞。 </li><li>挤在使用注射器的柱塞背面的细胞过滤剩余的组织。离心消化细胞以400×g离心在4℃下5分钟。小心取出上清液和悬浮颗粒在5毫升冰冷的FACS缓冲。算使用手动血球和台盼蓝染色来评估细胞的生存力的细胞。 </li></ol></li></ol><ol><li>重悬细胞沉淀到约1×10 6个细胞在200μl的FACS缓冲液中。 </li><li>根据制造商的方案中添加细胞生存力染料为死细胞排除。 </li><li>添加5微升的FcR阻断试剂的孵育在4℃下5分钟。 </li><li>添加的细胞表面的抗体抗CD45（2微升），谱系（CD3，CD20，CD56，CD66abce，和CD14;每个2微升），CD16（0.5微升），HLA-DR（3微升），的CD11c（5微升），CD123 （5微升），CD1C（3微升），和CD103（2微升），并孵育在4℃下15分钟。抗体上的细节可以在方法部分中找到，但在使用流式细胞仪滴定和优化抗体的精确的流量。洗去过量的抗体用PBS在400×g下5分钟。获取关于一个流动新鲜样品流式细胞仪或先于后来acquisit固定在1％多聚甲醛的细胞离子。 </li><li>确定在使用流式细胞仪测定18（ 图4）的流肺活检人DC。 注意:使用流式细胞仪分析软件的流，免疫细胞被门控CD45从其它肺细胞区分开来。死细胞可以排除，因为是积极为LIVE / DEAD染色细胞。所有活的 CD45 +细胞，细胞谱系细胞（B细胞，T细胞，NK细胞，中性粒细胞和单核细胞）的可使用针对CD20，CD3，CD56，CD66abce，CD14，和CD16抗体的鸡尾酒中一个信道被排除。以下即，HLA-DR +细胞将允许所有人DCs的识别。的MDC可以基于的CD11c的表达或分别缺少的CD11c的，的pdc加以区分。的MDC可以进一步分为CD1C +的MDC或CD141 +。 </li></ol>

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A., 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=Flow+Cytometric+Analysis+of+Myeloid+Cells+in+Human+Blood,+Bronchoalveolar+Lavage,+and+Lung+Tissues.">Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues.</a> Am J Respir Cell Mol Biol. , (2015).</li><li>Haniffa, M., 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=Human+tissues+contain+CD141hi+cross-presenting+dendritic+cells+with+functional+homology+to+mouse+CD103++nonlymphoid+dendritic+cells.">Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+ nonlymphoid dendritic cells.</a> Immunity. 37 (1), 60-73 (2012).</li><li>Britten, K. M., Howarth, P. H., Roche, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+on+resin+sections:+a+comparison+of+resin+embedding+techniques+for+small+mucosal+biopsies.">Immunohistochemistry on resin sections: a comparison of resin embedding techniques for small mucosal biopsies.</a> Biotech Histochem. 68 (5), 271-280 (1993).</li><li>Perfetto, S. P., Chattopadhyay, P. K., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seventeen-colour+flow+cytometry:+unravelling+the+immune+system.">Seventeen-colour flow cytometry: unravelling the immune system.</a> Nat Rev Immunol. 4 (8), 648-655 (2004).</li><li>Baharom, F., 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=Dendritic+Cells+and+Monocytes+with+Distinct+Inflammatory+Responses+Reside+in+Lung+Mucosa+of+Healthy+Humans.">Dendritic Cells and Monocytes with Distinct Inflammatory Responses Reside in Lung Mucosa of Healthy Humans.</a> J Immunol. 196 (11), 4498-4509 (2016).</li><li>Salvi, S., 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=Acute+inflammatory+responses+in+the+airways+and+peripheral+blood+after+short-term+exposure+to+diesel+exhaust+in+healthy+human+volunteers.">Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers.</a> Am J Respir Crit Care Med. 159 (3), 702-709 (1999).</li><li>Schon-Hegrad, M. A., Oliver, J., McMenamin, P. G., Holt, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+density,+distribution,+and+surface+phenotype+of+intraepithelial+class+II+major+histocompatibility+complex+antigen+(Ia)-bearing+dendritic+cells+(DC)+in+the+conducting+airways.">Studies on the density, distribution, and surface phenotype of intraepithelial class II major histocompatibility complex antigen (Ia)-bearing dendritic cells (DC) in the conducting airways.</a> J Exp Med. 173 (6), 1345-1356 (1991).</li><li>Saeys, Y., Gassen, S. V., Lambrecht, B. 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作者宣称，他们没有竞争的经济利益。

本文介绍了如何利用免疫组织化学生成健康人肺组织居民DC的详细的空间和表型鉴定和支气管镜检查期间收集支气管粘膜活检流式细胞仪。在以下各段中的协议的关键步骤中详细讨论。 关键步骤议定书切片和免疫组化:这是至关重要的，当不使用它们（步骤2.5），以保持活检块在-20℃。温馨块有时会变得柔软，不会一节为好。此外，保持块在-20℃将保留抗原?...

肺是在与外部环境的连续接触，并且高度暴露于既无害颗粒和微生物有能力引起的疾病。因此，它是免疫系统装入针对入侵病原体有效的免疫反应的关键，但要保持耐受吸入抗原不引起疾病是同样重要的。为了提供有效的免疫监视，呼吸系统衬有免疫细胞，包括树突细胞（DC）的网络。 DC是专业的抗原呈递细胞与激活幼稚T细胞的独特能力。在人的肺部，驻留的DC遇到抗原，然后进程并将其输送到肺引流淋巴结以供呈现给和活化的T细胞1，2，3的。 在人类的免疫系统，DCS可分为几个子集，具有次方CT但重叠的功能:CD1C +和CD141 +骨髓DC（的MDC）和CD123 +浆的DC（PDC上）4，5。而在人DCs最详细的知识从血液中的研究茎，现在明显的是，人体肺部也怀有DC亚群与T细胞刺激能力6，7，8，9的罕见群体。然而，积累的数据表明，免疫细胞，包括树突状，不同在它们的频率，表型，和功能取决于它们的解剖位置10。因此，为了研究免疫细胞从相关组织，以了解它们对局部免疫和容忍的贡献是重要的。总之，这强调需要解决肺部疾病时，研究肺居民的DC，尽管血液DC是更容易使用和访问<html>在人类。 在人类研究肺驻留的DC的首批研究采用免疫组织化学11，12，13主要依靠形态和单标记物，如HLA-DR和CD11c的表达，在组织切片。与此相反，最近的研究通常是在流式细胞依靠分析来研究不同的免疫细胞亚群。然而，由于很难找到唯一地标识特定的DC子集的单个细胞表面标记物，研究的潜在的限制施加仅四色流式细胞仪是包含具有类似表型标记如树突细胞群的风险。例如，CD11c的是在所有的骨髓DC和广大单核细胞表达。另一方面，在研究中采用更先进的流式细胞仪面板，从患者的外科手术切除非癌肺组织中通常使用外部参照"&gt; 10，14，15，16，虽然目前还不清楚这些稀有人群是否真正代表的DC存在于正常人。总体而言，研究在很大程度上是由于一个事实，即手术切除或整个人类的肺组织是稀缺的限制。 为了克服这些限制，这项工作介绍了如何执行空间分布及区议会在从谁接受支气管镜检查健康志愿者获得支气管粘膜活检表型鉴定的详细分析。几个小活检可以从每个单独的收集和随后可以嵌入用于切片和免疫组织化学或酶促消化先进流式细胞分析分析。在从支气管镜检查获得的支气管活检的形式使用的肺组织赋予使得能够执行ST的优点UDY上健康的志愿者，不像肺部开放手术，对于明显的原因，被限制为那些需要开胸手术的患者。此外，从健康志愿者支气管期间被取样的组织是生理上正常的，而相比之下，肺组织的非患部患者肺部疾病。另一方面，在活检小和检索单元的汇集几个活检即使当数，限制了可进行分析的类型。 虽然目前的工作重点是对DC，描述可以直接扩展的方法，包括其他（免疫）利益驻留在人体黏膜肺组织细胞。此外，该协议也直接适用于从肺部疾病，其中支气管镜检查是确立诊断，如慢性阻塞性肺疾病（COPD），结节病，或者肺癌的一部分的患者获得的样品。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Bronchoscopy&lt;/strong&gt;</td></tr><tr><td>Bronchoscope BF1T160</td><td>Olympus</td><td>BF1T160</td><td></td></tr><tr><td>Light source&amp;nbsp;</td><td>Olympus</td><td>Exera CV-160</td><td></td></tr><tr><td>Fenestrated forceps</td><td>Olympus</td><td>FB21C</td><td>Used to take biopsies</td></tr><tr><td>Bite Block</td><td>Conmed</td><td>1429</td><td>20 mm x 27 mm</td></tr><tr><td>Glucose 25%&amp;nbsp;</td><td></td><td></td><td>500 ml intravenous</td></tr><tr><td>Glycopyrronium bromide 0.2 mg/ml</td><td></td><td></td><td>Intravenous. Prevents mucus/saliva secretion</td></tr><tr><td>Mixt. Midazolam 1 mg/ml p.o</td><td></td><td></td><td>Can be used for extra relaxation</td></tr><tr><td>Lidocaine, 40 mg/ml</td><td></td><td></td><td>Mouth and throat administration / Gargled</td></tr><tr><td>Lidocaine 100 mg/ml spray</td><td></td><td></td><td>Administered to back of throat</td></tr><tr><td>Lidocaine 20 mg/ml spray</td><td></td><td></td><td>Administered via bronchoscope to airways</td></tr><tr><td colspan="4">&lt;strong&gt;GMA processing and embedding&lt;/strong&gt;</td></tr><tr><td>Glass vials</td><td></td><td></td><td>5 ml</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>32201-1L</td><td></td></tr><tr><td>Molecular sieves, 4 &amp;#197;</td><td>Alfa Aesar</td><td>88120</td><td>3-4 mm diameter pellets</td></tr><tr><td>Phenylmethylsulfonyl fluoride</td><td>Sigma-Aldrich</td><td>P-7626</td><td>0.035 g/100 ml acetone</td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I-6125</td><td>0.37 g/100 ml acetone</td></tr><tr><td>Polythene-flat&amp;nbsp; TAAB embedding capsules</td><td>TAAB laboratories</td><td>C094</td><td>500x 8 mm diameter, polythene, flat-bottom capsules</td></tr><tr><td>Capsule holder</td><td>TAAB laboratories</td><td>C054</td><td>Holds 25 8 mm capsules</td></tr><tr><td>JB-4 GMA embedding kit</td><td>Polysciences</td><td>00226</td><td>Contains JB-4 Solution A (0026A-800), JB-4 solution B (0026B-3.8), benzoyl peroxide (02618-12)</td></tr><tr><td>Methyl benzoate</td><td>Sigma-Aldrich</td><td>27614-1L</td><td></td></tr><tr><td>Silica gel with humidity indicator</td><td>Scharlau</td><td>GE0043</td><td>2.5-6 mm&amp;nbsp;</td></tr><tr><td colspan="4">&lt;strong&gt;GMA sectioning&lt;/strong&gt;</td></tr><tr><td>Glass microscope slides</td><td>ThermoFisher Scientific</td><td>10143562CEF</td><td>Cut edges, frosted end</td></tr><tr><td>Poly-L-Lysine solution</td><td>Sigma-Aldrich</td><td>P8920-500mL</td><td>1:10 for working solution</td></tr><tr><td>Sheet glass strips for ultramicrotomy</td><td>Alkar</td><td></td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P2287</td><td>Wash solution (0.1% Tween20)</td></tr><tr><td>LKB 7800B Knifemaker</td><td>LKB</td><td></td><td></td></tr><tr><td>Capsule splitter</td><td>TAAB laboratories</td><td>C065</td><td></td></tr><tr><td>Carbon steel single edge blades</td><td>TAAB laboratories</td><td>B054</td><td></td></tr><tr><td>Vice</td><td></td><td></td><td></td></tr><tr><td>Ammonia, 25%</td><td>VWR</td><td>1133.1000</td><td>2 ml in 1 L, 1:500 (0.05%)</td></tr><tr><td>Microtome</td><td>Leica</td><td></td><td>Leica RM 2165</td></tr><tr><td>Light source</td><td>Leica</td><td></td><td>Leica CLS 150 XE</td></tr><tr><td>Microscope with swing arm stand</td><td>Leica</td><td></td><td>Leica MZ6</td></tr><tr><td colspan="4">&lt;strong&gt;GMA Immunohistochemistry&lt;/strong&gt;</td></tr><tr><td>Diamond tipped pen</td><td>Histolab</td><td>5218</td><td></td></tr><tr><td>Hydrogen peroxide 30% solution</td><td>AnalaR Normapur</td><td>23619.264</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Tris</td><td>Roche</td><td>10708976001</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR chemicals</td><td>27810.295</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Millipore</td><td>82-045-2</td><td>Probumin BSA diagnostic grade</td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5546</td><td></td></tr><tr><td>Anti-human CD45 antibody</td><td>BioLegend</td><td>304002</td><td>Mouse monoclonal, clone HI30, isotype IgG1k. Working concentration of 500 ng/ml</td></tr><tr><td>Anti-human CD1a antibody</td><td>AbD Serotech</td><td>MCA80GA</td><td>Mouse monoclonal, clone NA1/34-HLK, isotype IgG2a. Working concentration of 10 &amp;micro;g/ml</td></tr><tr><td>Mouse monoclonal IgG1 isotype control</td><td>Abcam</td><td>ab27479</td><td></td></tr><tr><td>Mouse monoclonal IgG2a isotype control</td><td>Dako</td><td>X094301-2</td><td></td></tr><tr><td>Vectastain ABC Elite standard kit</td><td>Vector Labs</td><td>PK-6100</td><td></td></tr><tr><td>AEC (3-amino-9-ethylcarbazole) peroxidase substrate kite</td><td>Vector Labs</td><td>SK-4200</td><td></td></tr><tr><td>Mayers haematoxylin</td><td>HistoLab</td><td>01820</td><td></td></tr><tr><td>Permanent Aqueous Mounting Medium</td><td>AbD Serotech</td><td>BUF058C</td><td></td></tr><tr><td>Drying oven</td><td></td><td></td><td></td></tr><tr><td>DPX permanent mounting solution&amp;nbsp;</td><td>VWR</td><td>360292F</td><td></td></tr><tr><td>Light microscope</td><td>Leica</td><td></td><td>Leica DMLB</td></tr><tr><td>Microscope camera</td><td>Leica</td><td></td><td>Leica DFC 320</td></tr><tr><td>Analysis software</td><td>Leica</td><td></td><td>Leica Qwin V3</td></tr><tr><td colspan="4">&lt;strong&gt;Enzymatic digestion&lt;/strong&gt;</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Sigma-Aldrich</td><td>55021C</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>DTT-RO</td><td></td></tr><tr><td>Collagenase II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>10104159001&amp;nbsp;ROCHE</td><td></td></tr><tr><td>RPMI 1640</td><td>Sigma-Aldrich</td><td>R8758</td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Platform rocker</td><td>Grant instruments</td><td>PMR-30</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>14-432-22</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Flow cytometry&lt;/strong&gt;</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>LIVE/DEAD Aqua fixable dead cell stain kit</td><td>Life Technologies</td><td>L34957</td><td></td></tr><tr><td>CD45</td><td>BD</td><td>555485</td><td></td></tr><tr><td>CD3</td><td>BD</td><td>557757</td><td></td></tr><tr><td>CD20</td><td>BD</td><td>335829</td><td></td></tr><tr><td>CD56</td><td>Biolegend</td><td>318332</td><td></td></tr><tr><td>CD66abce</td><td>Miltenyi</td><td>130-101-132</td><td></td></tr><tr><td>HLA-DR</td><td>BD</td><td>555813</td><td></td></tr><tr><td>CD14</td><td>BD</td><td>557831</td><td></td></tr><tr><td>CD16</td><td>Biolegend</td><td>302026</td><td></td></tr><tr><td>CD11c</td><td>BD</td><td>560369</td><td></td></tr><tr><td>CD1c</td><td>Miltenyi</td><td>130-098-009</td><td></td></tr><tr><td>CD141</td><td>Miltenyi</td><td>130-090-514</td><td></td></tr><tr><td>CD103</td><td>Biolegend</td><td>350212</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>LSR II Flow cytometer</td><td>BD</td><td></td><td>Flow cytometer</td></tr><tr><td>FlowJo</td><td>FlowJo</td><td></td><td>Software for analysis</td></tr></tbody></table>

human lung dendritic cells: spatial distribution and phenotypic identification in endobronchial biopsies using immunohistochemistry and flow cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

研究表征人呼吸道组织驻留的免疫细胞，包括树突状，是有限的，这主要是由于这一事实，即外科手术切除或整个人肺组织是稀少。这里，从健康志愿者和开发的协议的支气管活检（EBB）获得的肺组织使用免疫组织化学研究中的组织中的免疫细胞或流式细胞术的微创方法进行了概述。 健康志愿者进行支气管镜检查，如前所述<s...

Watch this Scientific Journal Video about Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry at JoVE

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

human-lung-dendritic-cells-spatial-distribution-phenotypic

Research

JoVE Journal

Immunology and Infection

9.2K 次观看.  Karolinska Institutet. Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry. 

视频: Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

科研

免疫与感染

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

医学

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

使用琼脂糖包埋的振动切片机进行创新的肺部成像

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

作者聚焦:使用琼脂糖包埋的肺组织切片研究空间蛋白表达 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

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此视频中的章节

小鼠肺树突状细胞的分离

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

精密切割的小鼠肺切片可视化现场肺树突状细胞

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

评估对流感嗜血杆菌的呼吸道免疫应答

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片