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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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?...

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 border="0" cellpadding="0" cellspacing="0" width="1260">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bronchoscopy</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td style="width:628px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bronchoscope BF1T160</td>
			<td style="width:172px;">Olympus</td>
			<td style="width:148px;">BF1T160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light source&#160;</td>
			<td style="width:172px;">Olympus</td>
			<td style="width:148px;">Exera CV-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Fenestrated forceps</td>
			<td style="width:172px;">Olympus</td>
			<td style="width:148px;">FB21C</td>
			<td>Used to take biopsies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Bite Block</td>
			<td style="width:172px;">Conmed</td>
			<td style="width:148px;">1429</td>
			<td>20x27mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glucose 25%&#160;</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>500mL intravenous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glycopyrronium bromide 0.2mg/mL</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Intravenous. Prevents mucus/saliva secretion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mixt. Midazolam 1mg/mL p.o</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Can be used for extra relaxation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lidocaine, 40mg/mL</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Mouth and throat administration / Gargled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lidocaine 100mg/ml spray</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Administered to back of throat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lidocaine 20mg/ml spray</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>Administered via bronchoscope to airways</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GMA processing and embedding</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glass vials</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td>5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Acetone</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">32201-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Molecular sieves, 4A</td>
			<td style="width:172px;">Alfa Aesar</td>
			<td style="width:148px;">88120</td>
			<td>3-4mm diameter pellets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phenylmethylsulfonyl fluoride</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">P-7626</td>
			<td>0.035g/100ml acetone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Iodoacetamide</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">I-6125</td>
			<td>0.37g/100ml acetone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Polythene-flat&#160; TAAB embedding capsules</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">C094</td>
			<td>x500 8mm diameter, polythene, flat-bottom capsules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Capsule holder</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">C054</td>
			<td style="width:628px;">Holds 25 8mm capsules</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">JB-4 GMA embedding kit</td>
			<td style="width:172px;">Polysciences</td>
			<td style="width:148px;">00226</td>
			<td>Contains JB-4 Solution A (0026A-800), JB-4 solution B (0026B-3.8), benzoyl peroxide (02618-12)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Methyl benzoate</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">27614-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Silica gel with humidity indicator</td>
			<td style="width:172px;">Scharlau</td>
			<td style="width:148px;">GE0043</td>
			<td>2.5-6mm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GMA sectioning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Glass microscope slides</td>
			<td style="width:172px;">ThermoFisher Scientific</td>
			<td style="width:148px;">10143562CEF</td>
			<td>Cut edges, frosted end</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Poly-L-Lysine solution</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:148px;">P8920-500mL</td>
			<td>1:10 for working solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheet glass strips for ultramicrotomy</td>
			<td>Alkar</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P2287</td>
			<td>Wash solution (0.1% Tween20)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">LKB 7800B Knifemaker</td>
			<td>LKB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Capsule splitter</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">C065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Carbon steel single edge blades</td>
			<td style="width:172px;">TAAB laboratories</td>
			<td style="width:148px;">B054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Vice</td>
			<td style="width:172px;"></td>
			<td style="width:148px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Ammonia, 25%</td>
			<td style="width:172px;">VWR</td>
			<td style="width:148px;">1133.1000</td>
			<td>2mL in 1L, 1:500 (0.05%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Microtome</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica RM 2165</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light source</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica CLS 150 XE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Microscope with swing arm stand</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica MZ6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">GMA Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Diamond tipped pen</td>
			<td style="width:172px;">Histolab</td>
			<td style="width:148px;">5218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Hydrogen peroxide 30% solution</td>
			<td style="width:172px;">AnalaR Normapur</td>
			<td style="width:148px;">23619.264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S8032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Roche</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>VWR chemicals</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Millipore</td>
			<td>82-045-2</td>
			<td>Probumin BSA diagnostic grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonal IgG1 isotype control</td>
			<td>Abcam</td>
			<td>ab27479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse monoclonal IgG2a isotype control</td>
			<td>Dako</td>
			<td>X094301-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectastain ABC Elite standard kit</td>
			<td>Vector Labs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:312px;">AEC (3-amino-9-ethylcarbazole) peroxidase substrate kite</td>
			<td style="width:172px;">Vector Labs</td>
			<td style="width:148px;">SK-4200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Mayers haematoxylin</td>
			<td style="width:172px;">HistoLab</td>
			<td style="width:148px;">01820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Permanent Aqueous Mounting Medium</td>
			<td style="width:172px;">AbD Serotech</td>
			<td style="width:148px;">BUF058C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Drying oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">DPX permanent mounting solution&#160;</td>
			<td style="width:172px;">VWR</td>
			<td style="width:148px;">360292F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Light microscope</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica DMLB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Microscope camera</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica DFC 320</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Analysis software</td>
			<td style="width:172px;">Leica</td>
			<td></td>
			<td>Leica Qwin V3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Enzymatic digestion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>55021C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Dithiothreitol (DTT)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Collagenase II</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">DNase</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>10104159001&#160;ROCHE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">RPMI 1640</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Forceps</td>
			<td style="width:172px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Platform rocker</td>
			<td style="width:172px;">Grant instruments</td>
			<td>PMR-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">50 mL conical tubes</td>
			<td style="width:172px;">Falcon</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">40 &#181;m cell strainer</td>
			<td style="width:172px;">Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:148px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Flow cytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">LIVE/DEAD Aqua fixable dead cell stain kit</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD45</td>
			<td>BD</td>
			<td>555485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD3</td>
			<td>BD</td>
			<td>557757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD20</td>
			<td>BD</td>
			<td>335829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD56</td>
			<td>Biolegend</td>
			<td>318332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD66abce</td>
			<td>Miltenyi</td>
			<td>130-101-132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">HLA-DR</td>
			<td>BD</td>
			<td>555813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD14</td>
			<td>BD</td>
			<td>557831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD16</td>
			<td>Biolegend</td>
			<td>302026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD11c</td>
			<td>BD</td>
			<td>560369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD1c</td>
			<td>Miltenyi</td>
			<td>130-098-009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD141</td>
			<td>Miltenyi</td>
			<td>130-090-514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">CD103</td>
			<td>Biolegend</td>
			<td>350212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">LSR II Flow cytometer</td>
			<td>BD</td>
			<td></td>
			<td>Flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">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 ...

The main advantage of this technique is that a lot of information can be generated from small tissues obtained during bronchoscopy.This method can help answer key questions in mucosal immunology, where bronchoscopy is part of establishing the diagnosis, such as COPD, sarcoidosis, or lung cancer.Visual demonstration of this procedure will enable individuals new to this method to recognize when enzymatic digestion is complete.Demonstrating this procedure, along with Faezzah Baharom, will be Greg Rankin, a postdoc from our collaborator's lab at Ume

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

Research

JoVE Journal

Immunology and Infection

9.1K 次观看.  Karolinska Institutet. The main advantage of this technique is that a lot of information can be generated from small tissues obtained during bronchoscopy.This method can help answer key questions in mucosal immunology, where bronchoscopy is part of establishing the diagnosis, such as COPD, sarcoidosis, or lung cancer.Visual demonstration of this procedure will enable individuals new to this method to recognize when enzymatic digestion is complete.Demonstrating this procedure, along with Faezzah Baharom, will be Gre...

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

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

科研

免疫与感染

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 ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

通过成像流式细胞仪人单核细胞来源的树突状细胞的特征：两个单核细胞分离方法的比较

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...