我们感谢上海巴斯德医院流动细胞学设施、ABSL2设施和SPF动物设施的工作人员提供的技术帮助和建议。这项工作得到了以下资助：中国科学院战略重点研究计划（XDB29030103）、中国国家重点研发计划（2016YFA0502202）、中国国家自然科学基金委员会（31570886）。

动物实验得到了中国上海巴斯德学院动物护理和使用委员会的批准。所有实验都是根据机构动物护理和使用委员会批准的动物议定书进行的。
注：小鼠病毒感染和器官分离应在ABSL2条件下进行。
1. 接种PR8流感病毒和记录小鼠体重
<ol><li>准备8周大的雄性C57BL/6小鼠感染ABSL2室。 注：此协议也适用于雌性小鼠的实验。</li>
	<li>
PR8病毒的稀释：从-80°C冰柜中取出病毒，在冰上孵化，直到它融化成液体。将股票病毒彻底循环，用无菌磷酸盐缓冲盐水（PBS、135 mM NaCl、2.7 mM KCl、10 mM Na2HPO 4、1.8 mMKH 2 PO 4）在预冷1.5mL管中稀释病毒至2 PFU/μL。</li>
	<li>
小鼠麻醉： 称量每只小鼠，并计算鼠标重量（4倍（μL）的五巴比妥钠（2毫克/mL）的体积。在腹内注射五巴比妥钠的计算体积。 注：这一步骤是让小鼠稳定、平静地呼吸，以便准确滴答作响的病毒可以在体内接种。心跳过快或速度过慢表示麻醉不当。此外，建议使用兽医软膏，以避免眼干。</li>
	<li>
鼻内接种：彻底使稀释的PR8病毒漩涡。管道 10 μL，并仔细执行一侧一滴一滴的鼻内接种。在完成一个笼子中所有小鼠（最多5只小鼠）的接种后，在另一侧重复接种（通过接种保持小鼠的呼吸平静和稳定）。每只小鼠总共感染了40个PFU的PR8病毒。</li>
	<li>将老鼠放在温暖的笼子里，以便更好地恢复。</li>
	<li>每天监测鼠标重量10天。（感染日记录为第 0 天）。</li>
</ol>2. 从脾脏和介质淋巴结中分离淋巴细胞 （mLN）
<ol><li>
老鼠安乐死：把老鼠放在一个小房间里，从腔底和平地 泵入二氧化碳，对老鼠实施安乐死。当老鼠不动的时候，把老鼠拿出来，进行颈椎脱位，以确保老鼠完全死亡。用75%的乙醇浸入小鼠，并转移到生物安全罩。</li>
	<li>用解剖针将小鼠固定在吸和纸覆盖的解剖泡沫板上。用解剖剪刀切开腹部中线和后腿的皮肤，用钳子伸展皮肤。用解剖针固定拉伸的皮肤。</li>
	<li>为每只老鼠准备两个6厘米的菜，并把它们放在冰上。在每道菜中放入 70μm 细胞过滤器，并添加 5 毫升的 DMEM 补充 1% 胎儿牛血清 （DMEM （1% FBS））</li>
	<li>脾脏隔离：切开腹膜，用解剖剪刀露出腹腔。拿脾脏放在准备的菜里。</li>
	<li>mln 隔离： 将隔膜和笼肋骨底部切到胸腺附近。把肋骨拉到一边，用解剖针固定，露出胸腔。将肺拉到右侧，用钳子取mLN，在心脏下面和气管的腹侧附近。</li>
	<li>将mLN放入准备好的菜中。</li>
	<li>获得单细胞悬架：通过 70μm 细胞过滤器用 3 mL 注射器轻轻网格脾脏或 mLN。用1毫升新鲜DMEM（1%FBS）冲洗细胞过滤器。重新悬浮细胞悬架并转移到 15 mL 离心管中。</li>
	<li>离心细胞悬架在350 x g 6分钟在4°C。 删除超强剂并添加 1 毫升 DMEM （1% FBS）。</li>
	<li>用 1 mL 移液器彻底重新悬念细胞颗粒。将 4 mL 的 DMEM （1% FBS） 添加到脾脏细胞悬架中，并将其保留在冰上以进行以下操作。 注：首先，需要用1mL的介质（而不是5mL）重新悬息细胞颗粒，以完全隔离单细胞与颗粒。 从此以后，所有操作都可以在常规实验室中执行。</li>
	<li>
脾细胞计数
	<ol>
<li>通过将管子向上和向下转动数次来重新悬念细胞。将 10 μL 放入 90μL 的红血球 （RBC） 裂解缓冲区 （10 m M 特里斯 - Hcl pH 7.5， 155m NH4Cl）。在室温 （RT） 下孵化 3 分钟，并添加 900μL 的冷 PBS 以终止反应。</li>
		<li>离心机在400 x g 6分钟在4°C和删除超纳坦。用 100μL 的冷 PBS 重新悬念。将 10 μL 的细胞放入 10 μL 的 0.4% w/v 试盘蓝色中，并从混合物中取出 10 μL，用于使用血细胞计进行细胞计数。</li>
		<li>计算：将细胞计算为常规方法。简言之，在血小球仪上的两个对角形方块中计算细胞数，每个角方块获得 N1、N2。5mL细胞悬浮液的细胞浓度应计算为（N1+N2）/2 x 10 4/mL。</li>
	</ol>
</li>
</ol>3. PD-1和CXCR5多克隆Tfh细胞的免疫
<ol><li>染色与生物素抗CXCR5抗体。<ol>
<li>通过上下转动管子来重新悬浮细胞悬架。将 2 x 106 个细胞放入 FACS 管中，并添加 2 mL 的染色缓冲区（PBS（1% FBS，1 mM EDTA）。在涡流振荡装置上通过漩涡清洗。</li>
		<li>离心机在350 x g 6分钟在4°C。 倒出液体，将管口浸入吸水纸上两次，从而丢弃超纳坦。</li>
		<li>点击管子底部，用残留液体松开细胞颗粒。把管子放在冰上的管架上。 注：残留液体的体积约为25微升。</li>
		<li>为每根管子添加 0.2 μL 的反鼠标 CD16/CD32（Fc 受体阻滞剂）。通过轻轻敲击管底，在冰上孵育10分钟，使漩涡。 注：用每根管5μL的染色缓冲液稀释，为多个样品准备抗体混合物。混合物配方应通过稀释（n/10+1） x 0.2 μL Fc 受体阻滞剂放入 （n/10+1） x 5 μL 染色缓冲区，并在每个管子中加入 5.2 μL 混合物。</li>
		<li>通过点击管底，将 0.3 μL 的生物素防鼠标 CXCR5 添加到每个管和漩涡的污渍缓冲区残留物 30 μL 中。 注：准备步骤 3.1.4 中描述的混合物。</li>
		<li>在冰上孵育1小时，在30分钟内轻敲管子轻轻重新悬生细胞。 注：30 分钟的漩涡是为了避免细胞聚集物更好地染色。</li>
		<li>在漩涡振荡装置上添加2mL的染色缓冲器和漩涡。离心机在350 x g 6分钟，在4°C，并丢弃在步骤3.1.2描述的超纳坦。通过敲击管子并在冰上孵化以进行后续染色，从而产生漩涡。</li>
	</ol>
</li>
	<li>与其他表面标记染色。<ol>
<li>准备抗体混合物（表1）如步骤3.1.4所述。</li>
		<li>将抗体混合物加入每个管子中。通过敲击管底并在冰上孵育30分钟的漩涡。</li>
		<li>用2mL的染色缓冲区清洗电池。离心机在350 x g 6分钟在4°C。</li>
		<li>丢弃超纳坦，加入 400μL 的染色缓冲区。在涡旋振荡装置上对管子进行涡流，并将管子保持在黑暗中，直到进行流细胞学分析。</li>
	</ol>
</li>
</ol>4. PR8流感病毒NP特异性Tfh细胞的免疫化
注：这个染色NP特异性Tfh细胞的协议是从以前的研究15，17。
<ol><li>执行第 3.1 步中描述的生物素-CXCR5 染色，但用于染色的细胞数为 3 x 106， 以便有足够的抗原特异性细胞记录在流细胞学中。</li>
	<li>从 3.1.7 向管中添加 0.3 μL 的 APC 结合 IAbNP311-325 MHC II 类 （NP311-325）四重奏。为多个样品准备混合物，如步骤 3.1.4 注：在添加抗CD4抗体之前，必须先对四聚物进行污渍，因为CD4和抗CD4抗体之间的结合会干扰最佳的四聚体染色。</li>
	<li>轻轻敲击管子，在RT的黑暗中孵育30分钟，从而重新悬生细胞混合物。 注：在管子口盖上湿纸以减少蒸发</li>
	<li>加入其他表面标记（表1）的混合物，在RT继续孵育30分钟。</li>
	<li>洗涤和重新悬念细胞，如步骤 3.2.3 和 3.2.4 中描述的。</li>
</ol>5. 多克隆Tfh细胞中Bcl6的免疫化 
<ol><li>执行第 3 节中描述的表面标记 （表 2） 染色，但最后用 2 mL PBS 清洗，而不是染色缓冲区。</li>
	<li>离心机在350 x g 6分钟在4°C。 轻轻敲击管底，丢弃超纳坦和重新悬浮的细胞颗粒。</li>
	<li>将 300 μL 的 3.7% 甲醛溶液（用 PBS 稀释为 37% 甲醛）放入管中进行细胞固定。涡旋振荡装置上的漩涡，在RT孵育20分钟。</li>
	<li>在 4 °C 下以 500 x g 在 6 分钟内添加 2 mL 的染色缓冲区，用于清洗和离心机。 轻轻敲击管子，丢弃超纳坦和重新悬浮的细胞。</li>
	<li>添加 300 μL 的 0.2% Triton-X 100，并通过涡旋振荡装置上的漩涡重新悬浮细胞。在RT孵化15分钟。</li>
	<li>添加2 mL的染色缓冲区进行清洗。离心机在500 x g 6分钟在4°C。 轻轻敲击管底，丢弃超纳坦和悬浮细胞。</li>
	<li>为每根管子添加 1.5 μL 的 PE 抗 Bcl6 抗体。轻轻敲击管底以重新悬念混合物，并在 RT 孵育 2 小时，每 30 分钟轻轻敲击一次管子。 注意：在管子口盖上湿纸，以减少混合物蒸发。</li>
	<li>添加 2 mL 的 PBS 补充 0.01% 特里顿 X 100 到管子中。漩涡和离心机在500 x g 6分钟在4°C。</li>
	<li>作为步骤 5.8 重复洗涤。带 400μL 染色缓冲器的重新悬念细胞。在冰上保持细胞黑暗，直到流细胞学分析。</li>
</ol>6. IL21的细胞内染色
<ol><li>用PMA（磷12-肌酸13-醋酸盐）和离子霉素刺激细胞。<ol>
<li>以2 x 106 细胞从充血细胞悬浮和离心机在350 x g 6分钟在4°C。 用500μL的完整T细胞介质丢弃超纳坦和重新悬浮的细胞颗粒。将细胞转移到24井板中。</li>
		<li>将 20 nmol PMA 和 2 μmol 离子霉素添加到 500 μL 的完整介质18 中，并通过上下管道彻底混合。</li>
		<li>将步骤 6.2 中准备的解决方案添加到 24 井板中的单元格悬架中，并通过摇动板混合。通过在细胞中添加 500μL 完整 T 细胞介质而不添加 PMA 和离子霉素来设置未刺激的控制。在 37°C 的 CO2 孵化器中孵育 4 小时。</li>
		<li>在每口井中加入10微米BFA（布雷费尔丁A，用甲醇溶解），以阻止高尔吉仪器介于蛋白质运输。将板放回细胞孵化器，孵育2小时。</li>
	</ol>
</li>
	<li>执行细胞表面标记染色。<ol>
<li>通过轻轻上下管道将细胞转移到 FACS 管中来重新悬浮细胞。在管子中加入 1 mL 的染色缓冲液，在 4 °C 下以 350 x g 将离心机加入 6 分钟。</li>
		<li>执行 Fc 受体阻滞剂染色为步骤 3.1.4。</li>
		<li>执行细胞表面标记染色（表3），如步骤3.2.1至3.2.3，但用2 mL PBS清洗细胞除外。</li>
		<li>离心机在350 x g 6分钟在4°C。 通过点击管底丢弃超纳坦和重新悬浮的细胞。</li>
	</ol>
</li>
	<li>从活/死可修复水死细胞染色套件中加入 0.2 μL 试剂，并在 RT 的黑暗中孵育管 10 分钟，以执行死细胞的染色。</li>
	<li>在涡旋振荡装置的管子和漩涡中加入 2 mL 的 PBS。离心机在350 x g 6分钟在4°C和丢弃超纳坦。</li>
	<li>执行步骤 5.3 和 5.4 中描述的单元格固定。</li>
	<li>将 300μL 的染色缓冲器添加到重新悬架的电池中，并将管子存放在 4 °C 冰箱过夜。离心机在 500 x g 6 分钟在 4 °C 去除超纳坦。 注：此步骤可以省略，并继续紧跟第 6.5 步之后的步骤 6.7。</li>
	<li>在涡旋振荡装置上的管子和漩涡中加入 1 mL 的皂素缓冲区（用 0.2% （w/v） 皂素补充的染色缓冲区。在冰上孵化20分钟，进行细胞渗透。</li>
	<li>离心机在 500 x g 6 分钟在 4 °C 和丢弃超纳坦。</li>
	<li>在每个管子中加入0.5μL的人类Fc-IL21受体。将抗体混合物准备多次作为步骤 3.1.4，但用皂素缓冲剂而不是染色缓冲区稀释抗体除外。</li>
	<li>在 RT 孵化 1 小时，在 30 分钟内轻轻敲击管底以重新悬生细胞。</li>
	<li>加入2 mL的皂素缓冲液，以500 x g的500 x g清洗电池和离心机6分钟。丢弃超纳坦，重复清洗一次。</li>
	<li>在每个管子中添加 0.1 μL 的 APC 抗人 Ig （H+L）。作为步骤 3.1.4 为多个样品准备混合物，除非用皂素缓冲区而不是染色缓冲区稀释抗体。</li>
	<li>在冰上孵化样品30分钟，作为步骤6.11清洗。</li>
	<li>带 400μL 染色缓冲器的重新悬念细胞。将样品在冰上保持黑暗，直到流细胞学分析。</li>
</ol>7. GC B 和血浆细胞染色
<ol><li>以细胞为例，执行抗Fc受体抗体染色，从3.1.1到3.1.4。</li>
	<li>执行表面标记染色 （表 4）作为步骤从 3.1.5 到 3.1.7，但潜伏时间是 30 分钟而不是 1 小时。</li>
	<li>带 400μL 染色缓冲器的重新悬念细胞。将样品在冰上保持黑暗，直到流细胞学分析。</li>
</ol>8. 血清与血液的隔离
<ol><li>在感染后的第14天（d.p.i 14），从面部静脉收集血液，并将血液样本保存在4°C冰箱过夜。 注：在ABSL2条件下进行采血，从此以后，所有程序都可以在常规实验室进行。</li>
	<li>在4°C下将血液在400 x g 的离心机中抽吸10分钟。 用 200 μL 移液器小心隔离血清，以避免红细胞污染。每个样品分为 3 个管子，并存储在 -80°C。</li>
</ol>9. 与埃利萨一起检测 HA 特异性抗体滴度
<ol><li>每口井配有 50μL 的 2μg/mL HA 蛋白质溶液的涂层 ELISA 板，并在 4 °C 冰箱中孵育过夜。</li>
	<li>用 200 μL 的 PBS 稀释 0.05% 补间 （PBST） 洗三次。将 100 μL 的 PBST 稀释的 5% 脱脂牛奶加入每个油井，并在 RT 孵育 2 小时，以阻止非特异性结合。</li>
	<li>血清稀释和孵化：在PBS中准备3%的BSA作为稀释缓冲。稀释缓冲液中的血清稀释为 1：50、1：150、1：450。。。。。。至1：36450（建议进行3倍串行稀释）。将 50 μL 稀释的血清添加到每个油井中，并在 4°C 冰箱中孵育过夜。</li>
	<li>丢弃血清，通过在每个油井中加入 200 μL 的 PBST 快速洗一次井（轻轻摇动，然后丢弃）。然后用 200μL 的 PBST 缓慢地在摇床上清洗盘子，每次 5 分钟。</li>
	<li>添加 100 μL 的 HRP 标记的次要抗体，具体用于总 IgG、IgM、IgG1、IgG2b、IgG2c（1：5000，用 PBST 稀释），并在 RT 孵育 1 小时。按 9.4 中描述的 PBST 清洗板。</li>
	<li>从 4 °C 存储中取出等量的缓冲区 A 和缓冲区 B （TMB），并在使用前在 RT 预热至少 30 分钟。混合 A 和 B，在每口井中加入 100 μL TMB，并通过轻柔摇动在 RT 孵育 10-30 分钟。 注：这是TMB基板试剂集（BD，555214）手册的简要说明。</li>
	<li>派珀特 100 μL 的 2M H2SO4 进入每个井以终止反应。通过仪器阅读OD450值。</li>
	<li>数据分析：通过减去背景信号（空井的OD450值）获得最终的OD450值。绘制与每个样品的抗体同型对应的曲线，X 轴上的稀释因子和 Y 轴上的 OD450 值。</li>
</ol>10. 形而论
<ol><li>在d.p.i 10时隔离麻将。在 RT 中将甲醛溶液在 3.7% 中修复 1 小时。丢弃固定缓冲器，在摇床上用 PBS 清洗 5 分钟三次。</li>
	<li>在 4°C 下将 PBS 中的黄素（10% 蔗糖）脱水 1 小时，然后在 4°C 的 PBS 中脱水（30% 蔗糖），轻轻摇晃，直到松果沉入 15 mL 管的底部。</li>
	<li>取出脱水的孢子，嵌入最佳切割温度化合物和低温。</li>
	<li>在 -20°C 前冷却丙酮。 用预冷丙酮孵育组织部分10分钟。用PBS清洗组织三次。</li>
	<li>用 PBS 将含有 0.2% 三顿 X-100 的组织部分渗透 20 分钟，然后用 PBS 洗三次。</li>
	<li>在 RT 中用含有 10% 正常山羊血清（阻塞缓冲区）的 PBS 阻止非特定结合 1 小时，然后用 PBS 洗一次组织部分。</li>
	<li>用斯特里普塔维丁/生物素阻塞套件阻止非特定绑定。 注意：不要让样品从这一步开始干燥。</li>
	<li>与原发性抗体染色：小心地在组织部位添加阻塞缓冲稀释生物素-PNA（25μg/mL）和大鼠抗鼠IgD（2.5μg/mL）。在4°C冰柜的湿室中孵育组织部分过夜。</li>
	<li>用PBST快速清洗组织部分一次。快速洗涤PBST中的组织部分，缓慢摇晃5分钟。重复洗涤三次。</li>
	<li>稀释亚历克萨氟488-链球菌（1：500）和亚历克萨氟555-山羊抗鼠IgG（1：500）抗体与阻塞缓冲区，并将其添加到组织部分仔细</li>
	<li>在RT孵育1小时。</li>
	<li>将组织部分作为步骤 10.8 清洗，并小心地用延长溶液安装。小心地用盖片盖住组织，并在4°C时将其保持在黑暗中，直到进行粘膜分析。</li>
	<li>通过计算每个区域大小的 GC 数字来分析 GC 反应的大小。</li>
</ol>

<ol>
	<li>Johnston, R. 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=Bcl6+and+Blimp-1+are+reciprocal+and+antagonistic+regulators+of+T+follicular+helper+cell+differentiation.">Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.</a> Science. 325 (5943), 1006-1010 (2009).</li><li>Nurieva, R. I., 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=Bcl6+mediates+the+development+of+T+follicular+helper+cells.">Bcl6 mediates the development of T follicular helper cells.</a> Science. 325 (5943), 1001-1005 (2009).</li><li>Yu, D., 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=The+transcriptional+repressor+Bcl-6+directs+T+follicular+helper+cell+lineage+commitment.">The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.</a> Immunity. 31 (3), 457-468 (2009).</li><li>Pedros, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TRAF-like+motif+of+the+inducible+costimulator+ICOS+controls+development+of+germinal+center+TFH+cells+via+the+kinase+TBK1.">A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1.</a> Nature Immunology. 17 (7), 825-833 (2016).</li><li>Xu, H., 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=Follicular+T-helper+cell+recruitment+governed+by+bystander+B+cells+and+ICOS-driven+motility.">Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility.</a> Nature. 496 (7446), 523-527 (2013).</li><li>Lee, S. K., 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=B+cell+priming+for+extrafollicular+antibody+responses+requires+Bcl-6+expression+by+T+cells.">B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells.</a> The Journal of Experimental Medicine. 208 (7), 1377-1388 (2011).</li><li>Zotos, D., 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=IL-21+regulates+germinal+center+B+cell+differentiation+and+proliferation+through+a+B+cell-intrinsic+mechanism.">IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism.</a> The Journal of Experimental Medicine. 207 (2), 365-378 (2010).</li><li>Vogelzang, 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=A+fundamental+role+for+interleukin-21+in+the+generation+of+T+follicular+helper+cells.">A fundamental role for interleukin-21 in the generation of T follicular helper cells.</a> Immunity. 29 (1), 127-137 (2008).</li><li>Ansel, K. 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=A+chemokine-driven+positive+feedback+loop+organizes+lymphoid+follicles.">A chemokine-driven positive feedback loop organizes lymphoid follicles.</a> Nature. 406 (6793), 309-314 (2000).</li><li>Shi, 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=PD-1+Controls+Follicular+T+Helper+Cell+Positioning+and+Function.">PD-1 Controls Follicular T Helper Cell Positioning and Function.</a> Immunity. 49 (2), 264-274 (2018).</li><li>Methot, S. P., Di Noia, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Somatic+Hypermutation+and+Class+Switch+Recombination.">Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.</a> Advanced ImmunoChemical. 133, 37-87 (2017).</li><li>Crotty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+helper+CD4+T+cells+(TFH).">Follicular helper CD4 T cells (TFH).</a> Annual Review of Immunology. 29, 621-663 (2011).</li><li>Calame, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cells:+finding+new+light+at+the+end+of+B+cell+development.">Plasma cells: finding new light at the end of B cell development.</a> Nature Immunology. 2 (12), 1103-1108 (2001).</li><li>Yoo, J. K., Fish, E. N., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAPCs+promote+follicular+helper+T+cell+differentiation+of+Ag-primed+CD4++T+cells+during+respiratory+virus+infection.">LAPCs promote follicular helper T cell differentiation of Ag-primed CD4+ T cells during respiratory virus infection.</a> The Journal of Experimental Medicine. 209 (10), 1853-1867 (2012).</li><li>Leon, B., Bradley, J. E., Lund, F. E., Randall, T. D., Ballesteros-Tato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP3++regulatory+T+cells+promote+influenza-specific+Tfh+responses+by+controlling+IL-2+availability.">FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability.</a> Nature Communications. 5, 3495 (2014).</li><li>He, L., 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=Extracellular+matrix+protein+1+promotes+follicular+helper+T+cell+differentiation+and+antibody+production.">Extracellular matrix protein 1 promotes follicular helper T cell differentiation and antibody production.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (34), 8621-8626 (2018).</li><li>Le&#243;n, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+TH2+development+by+CXCR5++dendritic+cells+and+lymphotoxin-expressing+B+cells.">Regulation of TH2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells.</a> Nature Immunology. 13 (7), 681-690 (2012).</li><li>Wang, H., 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=The+transcription+factor+Foxp1+is+a+critical+negative+regulator+of+the+differentiation+of+follicular+helper+T+cells.">The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells.</a> Nature Immunology. 15 (7), 667-675 (2014).</li><li>Bouvier, N. M., Lowen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Influenza+Virus+Pathogenesis+and+Transmission.">Animal Models for Influenza Virus Pathogenesis and Transmission.</a> Viruses. 2 (8), 1530-1563 (2010).</li><li>Miyauchi, K., 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=Protective+neutralizing+influenza+antibody+response+in+the+absence+of+T+follicular+helper+cells.">Protective neutralizing influenza antibody response in the absence of T follicular helper cells.</a> Nature Immunology. 17 (12), 1447-1458 (2016).</li><li>Rodriguez, L., Nogales, A., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+Virus+Studies+in+a+Mouse+Model+of+Infection.">Influenza A Virus Studies in a Mouse Model of Infection.</a> Journal of Visualized Experiments. (127), (2017).</li><li>Kitano, 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=Bcl6+protein+expression+shapes+pre-germinal+center+B+cell+dynamics+and+follicular+helper+T+cell+heterogeneity.">Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.</a> Immunity. 34 (6), 961-972 (2011).</li><li>Yusuf, I., 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=Germinal+center+T+follicular+helper+cell+IL-4+production+is+dependent+on+signaling+lymphocytic+activation+molecule+receptor+(CD150).">Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150).</a> The Journal of Immunology. 185 (1), 190-202 (2010).</li><li>Sun, J., Dodd, H., Moser, E. K., Sharma, R., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+cell+help+and+innate-derived+IL-27+induce+Blimp-1-dependent+IL-10+production+by+antiviral+CTLs.">CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs.</a> Nature Immunology. 12 (4), 327-334 (2011).</li></ol>

作者没有什么可透露的。

由于在提供B细胞帮助产生高亲和力抗体方面具有特殊作用，Tbh细胞在分化和操纵机制方面进行了广泛的研究，为疫苗设计提供了新的策略。流感病毒感染诱发强大的Tfh和GC B细胞反应，因此是这一研究领域的一个适当模型。本文通过鼻内接种、流细胞学、免疫荧光和ELISA对Tfh相关反应的评价，描述了流感病毒感染的治疗方案。这些检测将有助于检测Tfh分化、GC B发育和流感病毒特异性抗体，并帮助?...

近十年来，许多研究都集中在新发现的CD4+T细胞子集Tfh细胞子集上，因为它在胚芽中心（GC）B发育中的重要作用。B细胞淋巴瘤6（Bcl6），主要被认为是一种基因抑制剂，是Tfh细胞的血统定义因子，以证明Bcl6的异位表达足以驱动Tfh分化，而Bcl6的缺乏导致Tfh分化1，2，3消失。与其他CD4+T助剂子集通过迁移到炎症部位来发挥其作用或功能不同，Tfh细胞主要在脾脏和淋巴结的B细胞卵泡区提供B细胞帮助。共刺激分子 ICOS 和 CD40L 在 Tfh 和 GC B 细胞之间的相互作用中起着重要作用。在Tfh分化过程中，ICOS从认知B细胞传输必要的信号，并充当接收来自旁观者B细胞的迁移信号的受体，用于B细胞区域定位4、5。CD40L是来自Tfh细胞的信号的调停器，用于B细胞增殖和生存6。与CD40L扮演类似角色的另一个因素是细胞因子IL21，它主要由Tfh细胞分泌。IL21直接调节GC B细胞的高亲和力抗体的开发和生产，但它在Tfh分化中的作用仍有争议。PD-1 和 CXCR5 现在最常用于识别流细胞学分析中的 Tfh 细胞，它们也在这个子集的分化和功能中发挥着重要作用。CXCR5是B细胞卵泡化学素的受体，并调解Tbh细胞在B细胞卵泡9的本地化。PD-1现已确定不仅具有卵泡导引功能，而且在GC B细胞亲和力成熟10的过程中传输临界信号。基于这些发现，评估这些分子的表达基本上可以反映Tfh细胞的成熟和功能。
GC 是继发淋巴器官中的诱导瞬态微解剖结构，高度依赖 Tfh 细胞，因此是评估 Tfh 反应的完美读出。在GC中，在接收细胞因子和共刺激分子调停的信号后，B细胞受到类开关和体细胞多造，以产生高亲和力抗体11。差异抗体类切换发生在差异细胞因子利基，其中IL4和IL21诱导IgG1类切换，而IFNγ诱导IgG2类开关12。等离子体细胞是分泌抗体的产生者，是最终分化的细胞。与Tfh细胞一样，GC中B细胞的发展与许多重要分子的动态表达有关。根据目前的研究，GC B细胞可以识别为B220+PNA+Fas+或B220+GL7+Fas+细胞和等离子体细胞，与其前体相比，B220的低调节表达和向上调节CD138表达13。此外，这两种特征都可以在流动细胞学和免疫荧光分析中检测出来，从而对GC反应进行适当的评估。
在流感病毒感染中诱导强大的细胞和幽默反应，Tfh和Th1细胞主导CD4+T细胞反应14，这使得它成为Tfh细胞分化研究的完美模型。甲型流感/波多黎各/8/34 H1N1（PR8），这是一种常用的适应鼠标的菌株，在这项研究中经常使用14，15，16。在这里，我们描述了流感病毒感染中Tfh研究相关检测的一些基本方案：1）PR8病毒的鼻内接种：2） 抗原特异性Tfh细胞、GC B和血浆细胞以及IL21检测与流细胞学：3） GC的组织学可视化：4） 检测抗原特异性抗体滴度在血清与 ELISA.这些协议为与Tfh相关的研究中的新研究人员提供了必要的技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Immunostaining of Tfh cells, NP-specific Tfh cells and Bcl-6</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Sigma</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD16/32 mouse</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-0161-86</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated-IAbNP311-325 MHC class II tetramer</td>
			<td>NIH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bcl-6 PE</td>
			<td>Biolegend</td>
			<td>358504</td>
			<td>clone:7D1</td>
		</tr>
		<tr>
			<td>Biotin-CXCR5</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-7185-82</td>
			<td>clone: SPRCL5</td>
		</tr>
		<tr>
			<td>CD4 Percp-eFluor 710</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-0041-82</td>
			<td>clone:GK1.5</td>
		</tr>
		<tr>
			<td>CD44 eVolve 605</td>
			<td>Thermo Fisher Scientifi</td>
			<td>83-0441-42</td>
			<td>clone:IM7</td>
		</tr>
		<tr>
			<td>CD44 FITC</td>
			<td>Thermo Fisher Scientifi</td>
			<td>11-0441-82</td>
			<td>clone:IM7</td>
		</tr>
		<tr>
			<td>CD62L FITC</td>
			<td>BD Pharmingen</td>
			<td>553150</td>
			<td>clone:MEL-14</td>
		</tr>
		<tr>
			<td>ICOS BV421</td>
			<td>Biolegend</td>
			<td>564070</td>
			<td>clone:7E.17G9</td>
		</tr>
		<tr>
			<td>PD1 PE/Cy7</td>
			<td>Biolegend</td>
			<td>135216</td>
			<td>clone:29F.1A12</td>
		</tr>
		<tr>
			<td>Streptavidin BV421</td>
			<td>BD Pharmingen</td>
			<td>563259</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin PE</td>
			<td>BD Pharmingen</td>
			<td>554081</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracelluar staining of IL21</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37% formaldehyde</td>
			<td>Sigma</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human IgG</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>109-605-098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A</td>
			<td>Sigma</td>
			<td>B6542</td>
			<td></td>
		</tr>
		<tr>
			<td>human FCc IL-21 receptor</td>
			<td>R&#38;D System</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ionomycin</td>
			<td>Sigma</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Fixable Aqua Dead Cell staining kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>MP</td>
			<td>102855</td>
			<td></td>
		</tr>
		<tr>
			<td>GC B and plasma cells staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B220 APC</td>
			<td>Thermo Fisher Scientific</td>
			<td>17-0452-81</td>
			<td>clone:RA3-6B2</td>
		</tr>
		<tr>
			<td>CD138 PE</td>
			<td>BD Pharmingen</td>
			<td>561070</td>
			<td>clone:281-2</td>
		</tr>
		<tr>
			<td>CD95 (FAS) PE/Cy7</td>
			<td>BD Pharmingen</td>
			<td>557653</td>
			<td>clone:Jo2</td>
		</tr>
		<tr>
			<td>IgD eFluor 450</td>
			<td>Thermo Fisher Scientific</td>
			<td>48-5993-82</td>
			<td>clone:11-26c</td>
		</tr>
		<tr>
			<td>PNA FITC</td>
			<td>Sigma</td>
			<td>L7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay of HA-specific antibody titer with ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PR8-HA</td>
			<td>Sino Biological</td>
			<td>11684-V08H</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SSBC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse Ig (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgM (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgG1 (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgG2b (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti mouse IgG2c (SBA Clonotyping System-HRP)</td>
			<td>SouthernBiotech</td>
			<td>5300-05</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB Substrate Reagent Set</td>
			<td>BD Pharmingen</td>
			<td>555214</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555-Goat-anti rat IgG</td>
			<td>Life Technology</td>
			<td>A21434</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgD</td>
			<td>Biolegend</td>
			<td>405702</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated PNA</td>
			<td>Vector laboratories</td>
			<td>B-1075</td>
			<td></td>
		</tr>
		<tr>
			<td>dilute Alexa Fluor 488-streptavidin</td>
			<td>Life Technology</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>SouthernBiotech</td>
			<td>0060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pro-long gold antifade reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3630</td>
			<td></td>
		</tr>
		<tr>
			<td>STREPTAVIDIN/BIOTIN blocking kit</td>
			<td>Vector laboratories</td>
			<td>SP-2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of t follicular helper cells and germinal center response during influenza a virus infection in mice

T 卵泡帮助器 （Tfh） 细胞是一个独立的 CD4+ T 细胞子集，专门为细菌中心 （GC） 的发育和高亲和力抗体的生成提供帮助。在流感病毒感染中，诱导强大的Tfh和GC B细胞反应，以促进有效的病毒根除，这为Tfh相关研究提供了合格的鼠标模型。在本文中，我们描述了在小鼠感染流感病毒期间检测与Tfh相关的基本免疫反应的协议。这些协议包括：流感病毒的鼻内接种：流细胞染色和多克隆和抗原特异性Tfh细胞、GC B细胞和血浆细胞的分析：GC的免疫荧光检测;血清中流感病毒特异性抗体的酶相关免疫体检测（ELISA）。这些检测方法基本量化了Tfh细胞在流感病毒感染中的分化和功能，从而有助于研究区分机制和操作策略。

流感病毒感染中小鼠发病率的描述 流感病毒感染后，小鼠因疾病而不太活跃和厌食，这反映在严重的体重减轻上，这是监测小鼠发病率的常用症状19。如 图1a所示，PR8病毒感染的小鼠在第6天开始减肥，第8天达到最高损失水平，10日恢复到初始水平。不出所料，在PBS治疗的对照小鼠中，整个期间都没有观察到减肥。对于体内症状?...

Watch this Scientific Journal Video about Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice at JoVE.com

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

本文描述了在流感病毒感染小鼠模型中评估Tfh和GC B反应的协议。

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

evaluation-t-follicular-helper-cells-germinal-center-response-during

Research

JoVE Journal

Immunology and Infection

5.1K 次观看.  University of Chinese Academy of Sciences. 本文描述了在流感病毒感染小鼠模型中评估Tfh和GC B反应的协议。

视频: Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...