这项工作得到了NIH拨款P20 GM109098和从普拉斯佩罗到埃德温·万的创新奖计划的支持。流式细胞术实验在WVU流式细胞术和单细胞核心设施中进行，该设施得到了NIH授予S10 OD016165，U57 GM104942，P30 GM103488和P20 GM103434的支持。

所有执行的协议和程序都得到了西弗吉尼亚大学机构动物护理和使用委员会（IACUC）的批准。小鼠被安置在西弗吉尼亚大学动物馆的无病原体条件下。
1. 溶液的制备
<ol><li>制备灌注缓冲液（磷酸盐缓冲盐水，PBS）。每只小鼠使用大约两个 10 mL 等分试样的冰冷 PBS。</li>
	<li>准备肺细胞培养基/流式细胞系统缓冲液。FACS缓冲液含有补充1%胎牛血清（FBS）的PBS。在肺切除和转移的整个过程中保持培养基的低温。每个肺样本准备约 8 mL。在实验前准备新鲜的培养基/ FACS缓冲液。</li>
	<li>制备用于单细胞分离的解离缓冲液。缓冲液在汉克缓冲盐溶液（HBSS）中含有1毫克/毫升胶原酶D和200微克/毫升DNase I。在实验前从储备溶液（100mg / mL胶原酶D和10mg / mL DNase I）中新鲜制备。每个肺样本大约需要 6 mL。使用前确保缓冲液达到室温。</li>
	<li>制备用于肺组织均质化的均质化缓冲液。缓冲液含有PBS和1x蛋白酶和磷酸酶抑制剂鸡尾酒（储备= 100x）。在实验前新鲜准备。在整个均质化过程中保持缓冲液的低温。大约200μL缓冲液足以使肺的单个右叶匀浆化。</li>
</ol>2. 短暂性脑中动脉阻塞
注意:tMCAO通过单丝插入大脑中动脉的程序在之前有详细的记录10。在这些实验中，使用了8至12周龄的雄性C57BL / 6J小鼠，重25-30g。
<ol><li>简而言之，在手术前皮下给予小鼠2mg / kg美洛昔康。 使用5%异氟醚在诱导室中深度麻醉小鼠。使用脚趾捏方法确认深度麻醉。在使用鼻锥的手术过程中用1-2%异氟醚维持麻醉。在整个过程中，通过在小鼠下方放置温暖的毯子，将体温保持在36.5 - 38°C。所有手术工具应在手术前一天进行高压灭菌。</li>
	<li>使用70%乙醇剃须并准备腹侧颈部的皮肤，然后进行氯己定磨砂。在进行第一个切口之前，皮下注射2mg / kg布比卡因在切口区域。使用眼药膏遮住眼睛，防止眼睛干燥。做一个中线颈部切口。轻轻拉开气管周围的软组织。</li>
	<li>识别左颈总动脉和颈外动脉。</li>
	<li>将临时缝合线（6/0号）应用于颈总动脉以阻止血液流动。在颈外动脉做一个切口。</li>
	<li>将硅橡胶涂层的单丝（大小为6-0）插入颈外动脉，然后将单丝推进到大脑中动脉。将单丝留在大脑中动脉中60分钟（闭塞）。</li>
	<li>使用激光多普勒流量计监测血流速率。流向大脑中动脉的流速降低> 80% 表明闭塞成功。</li>
	<li>闭塞60分钟后，取出单丝以允许再灌注发生。关闭切口。</li>
	<li>在假手术小鼠中，在不插入单丝的情况下执行步骤2.1-2.6。在这些小鼠中，血液流动速率应在整个过程中保持稳定。</li>
	<li>每天监测小鼠并使用标准评分标准11测量神经缺陷。0 – 无神经功能障碍;1 – 当被尾巴抬起时缩回对侧前爪;2-当被尾巴抬起时，向对侧旋转;3-走路时跌落到对侧;4 – 不自发行走或昏迷;5 – 死亡。</li>
	<li>术后每24小时皮下注射生理盐水和镇痛药（美洛昔康，2mg / kg），持续3天。</li>
	<li>在tmCAO后24和72小时分离肺部以进行下游分析。</li>
</ol>3. 收获肺组织
<ol><li>通过腹膜内（ip）注射100mg / kg氯胺酮和10mg / kg木乃静嗪来深度麻醉小鼠。使用脚趾捏方法确认深度麻醉。</li>
	<li>为了进行全身心经灌注12，将小鼠固定在手术平台上并用70%乙醇润湿毛皮以减少皮毛进入体腔的分布。使用精细的夹层剪刀在尾腹部做一个横向切口，然后在腹膜的中线切口，在穿刺膈肌之前停止在胸腔处。<ol><li>不要穿过肌肉/隐藏进入腹膜腔，也不要继续切口经过腹部。</li>
	</ol></li>
	<li>一旦到达胸腔，在刺穿隔膜之前立即停止切口。注意不要损害心脏和肺部，这可能会损害灌注质量或细胞恢复</li>
	<li>使用精细的夹层剪刀使腹膜的中线切口在刺穿隔膜之前停止在胸腔处。</li>
	<li>使用镊子抓住胸骨远端的鼠标，向上抬起，同时穿过横膈膜，确保不会损害心脏，肺或脉管系统。一旦器官可见，就通过肋骨笼做一个切口，分离并固定在肋骨壳上，使心脏和肺部完全暴露。</li>
	<li>小心地在主动脉弓附近的右心房做一个小切口。这将作为灌注的流出。</li>
	<li>紧接着，将连接到装有10 mL冷PBS的注射器上的25 G x 5/8针轻轻插入左心室远端上表面。注意不要通过心室间隔插入右心室。针头不应插入左心室。 注意:如果心室隔被刺穿，液体将从小鼠的鼻子中涌出。这将降低灌注质量并导致细胞损失。</li>
	<li>如果需要，可以使用夹具在灌注过程中将针头牢固地固定在左心室中。</li>
	<li>稳定但轻轻地将 10 mL 冷 PBS 推入心脏。当液体从右心房排出时，小鼠组织应开始灌注并清除血液。<ol><li>推入第二等分试样的 10 mL PBS，直到发生足够的血液清除。肝脏将具有浅棕色的外观，肺部将从红粉红色过渡到大部分白色。</li>
	</ol></li>
	<li>使用镊子和细切剪刀，小心地去除所有周围组织，包括心脏，气管，食道，胸腺，结缔组织，淋巴结节和大支气管。</li>
	<li>分离单个肺叶，并将肺叶放入含有1-2 mL等分试样的冷肺细胞培养基的冰上的培养皿中。</li>
</ol>4. 使用磁珠均质机对多重磁珠阵列的肺组织进行均质化
<ol><li>预冷装有 3 个无菌 2.3 mm 氧化锆/二氧化硅珠的 2 mL 锥形螺旋盖管。向管中加入200μL冷均质缓冲液。</li>
	<li>称量转子，将转子转移到含有磁珠和均质缓冲液的预冷锥形管中。 注意:在200μL缓冲液中匀浆的约50-100mg组织将足以检测样品中的细胞因子和趋化因子表达。<ol><li>使用基于磁珠的均质机（参见 材料表）在4，000rpm下均质转子2分钟。</li>
		<li>确保组织完全均质化。如果需要，请增加时间。</li>
	</ol></li>
	<li>均质化后，将管在预冷（4°C）微量离心机中以15，870 ×g 离心3分钟。</li>
	<li>将上清液转移到预冷的1.5mL微量管中。</li>
	<li>将样品直接放在冰上立即使用，或将样品储存在-80°C以备将来使用。避免多次冻融循环。</li>
</ol>5. 肺组织解离和单细胞分离
<ol><li>将肺细胞培养基倒掉剩余的肺叶，以避免解离缓冲液的稀释。然后，小心地将含有1mL解离缓冲液的25G和5/8针头的1 mL注射器插入肺组织中。</li>
	<li>将解离缓冲液的小份（约1/4至 1/5）注入每个肺叶并轻轻充气。 注意:大部分缓冲液在充气后不久就会冲出，因为肺叶已被切除。</li>
	<li>重复注入解离缓冲液1-2x。</li>
	<li>在室温下用解离缓冲液孵育肺叶2分钟。</li>
	<li>用细的解剖剪刀将肺叶切成小块。</li>
	<li>将切碎的肺片与解离缓冲液转移到15mL离心管中。 补充额外的解离缓冲液，共6 mL。剧烈涡旋1分钟。</li>
	<li>将样品在37°C下孵育45分钟。 每7-8分钟剧烈涡旋一次。</li>
	<li>孵育45分钟后，剧烈涡旋样品1分钟以获得最佳结果。</li>
	<li>通过100μm细胞过滤器进一步解离样品，以除去残留的，不需要的结缔组织和间质组织。</li>
	<li>将样品以380 ×g离心10分钟，弃去上清液。</li>
	<li>向样品中加入5 mL冷肺细胞培养基。通过温和的涡旋重悬细胞沉淀。</li>
	<li>将样品以380 ×g离心10分钟，弃去上清液。</li>
	<li>加入1 mL冷肺细胞培养基。通过温和的涡旋重悬细胞沉淀。</li>
	<li>通过100μm细胞过滤器，并计数细胞。</li>
</ol>6. 肺免疫细胞生态位的流式细胞术分析
<ol><li>将1-2×106 个细胞/抗体集放入96孔圆底板（共3组）中。以830 ×g离心细胞3分钟。弃去上清液。</li>
	<li>将细胞沉淀重悬于50μL含有可固定活/死污渍的冷PBS中。根据制造商的说明准备污渍。</li>
	<li>将细胞在4°C下孵育20分钟，避光。以830 ×g离心细胞3分钟。弃去上清液。</li>
	<li>用含有5μg/ mL抗小鼠CD16 / 32抗体的25μL冷FACS缓冲液重悬细胞沉淀。在4°C下孵育10-15分钟。</li>
	<li>向细胞中加入25μL含有 表1 中列出的抗体组合的冷FACS缓冲液。通过轻柔移液混合。 注意:用于抗体染色的FACS缓冲液的总体积为50μL。因此，在制备25μL抗体预混液时，抗体的浓度应为最终浓度的两倍。</li>
	<li>将细胞在4°C下孵育20分钟，避光。以830 ×g离心细胞3分钟。弃去上清液。</li>
	<li>用100μL冷FACS缓冲液重悬细胞。以830 ×g离心细胞3分钟。弃去上清液。</li>
	<li>用100μL冷FACS缓冲液重悬细胞。转移到含有150μL额外FAC缓冲液的聚苯乙烯圆底5 mL FACS管中。立即使用流式细胞仪分析样品。 注意:使用以下步骤进行细胞固定可以在以后分析样品。</li>
	<li>在步骤6.7.之后，用100μL1%多聚甲醛重悬样品在PBS中。</li>
	<li>将样品在4°C下孵育15分钟，避光。以830 ×g离心细胞3分钟。弃去上清液。</li>
	<li>用100μL冷FACS缓冲液重悬细胞。以830 ×g离心细胞3分钟。弃去上清液。</li>
	<li>用100μL冷FACS缓冲液重悬细胞。将细胞储存在4°C，避光。在72小时内分析样品。</li>
</ol>7. 用于细胞因子和趋化因子检测的多重磁珠阵列
注意:根据制造商方案，使用市售的促炎趋化因子和炎症多重组合（见 材料表）来确定tmCAO诱导后肺部趋化因子和细胞因子的表达，该方案已详细描述13。
<ol><li>简而言之，如果在组织均质化后将分析物储存在-80°C，则在冰上解冻分析物。使用前在4°C下以590 ×g 离心样品2分钟以除去任何残留组织。</li>
	<li>通过将已知浓度为10 ng/ mL的标准鸡尾酒（C7）连续稀释到7个标准品（C1-6）中，最后一个标准品单独是测定缓冲液（C0），从而生成标准曲线。</li>
	<li>一旦所有试剂都升温到室温，将25μL每个标准品和25μL测定缓冲液加入V底96孔板中。</li>
	<li>向每个样品孔中，向V底96孔板中加入25 μL肺匀浆物（分析物）和25 μL测定缓冲液</li>
	<li>用力涡旋预包被的微球，然后将25μL预包衣珠加入每个标准品和样品孔中。</li>
	<li>密封板，并用铝箔覆盖物避光。在室温下以110rpm摇动板2小时。</li>
	<li>以230 ×g 离心5分钟。在1x下加入200μL洗涤缓冲液（储备溶液为20x，用水稀释至1x）。孵育1分钟。</li>
	<li>以230 ×g 离心5分钟。用25μL检测抗体重悬标准品和样品。</li>
	<li>密封并盖住板，以防光线。在室温下以110rpm摇动板1小时。</li>
	<li>向每个标准品中加入25μL链霉亲和素 - 藻红素（SA-PE）并充分采样。</li>
	<li>密封并盖住板，以防光线。在室温下以110rpm摇动30分钟。</li>
	<li>将板以230 x g 旋转5分钟。将标准品和样品重悬于1x洗涤缓冲液中。</li>
	<li>转移到标记良好的聚苯乙烯圆底5 mL FACS管中，该管含有额外的150μL 1x洗涤缓冲液。</li>
	<li>使用流式细胞仪测定标准品和样品的PE信号荧光强度。</li>
	<li>使用PE的平均荧光强度（MFI）与预定浓度的对比，为每个趋化因子和细胞因子构建标准曲线。</li>
	<li>使用标准曲线和每个样品的MFI确定每种趋化因子和细胞因子的浓度。多重检测面板提供数据分析软件，可用于确定每种分析物的浓度。顶叶/上叶的重量用于确定每毫克组织中细胞因子或趋化因子的浓度。</li>
</ol>

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S., Zhao, A., Sun, B., Jiang, W., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Cytokine+Profiling+of+Stimulated+Mouse+Splenocytes+Using+a+Cytometric+Bead-based+Immunoassay+Platform.">Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform.</a> Journal of Visualized Experiments. (129), e56440 (2017).</li><li>Shi, C., Pamer, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+recruitment+during+infection+and+inflammation.">Monocyte recruitment during infection and inflammation.</a> Nature Reviews Immunology. 11 (11), 762-774 (2011).</li><li>Monaghan, K. L., Zheng, W., Hu, G., Wan, E. C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+and+Monocyte-Derived+Antigen-Presenting+Cells+Have+Distinct+Gene+Signatures+in+Experimental+Model+of+Multiple+Sclerosis.">Monocytes and Monocyte-Derived Antigen-Presenting Cells Have Distinct Gene Signatures in Experimental Model of Multiple Sclerosis.</a> Frontiers in Immunology. 10, 2779 (2019).</li><li>Zhai, X., 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+novel+technique+to+prepare+a+single+cell+suspension+of+isolated+quiescent+human+hepatic+stellate+cells.">A novel technique to prepare a single cell suspension of isolated quiescent human hepatic stellate cells.</a> Science Reports. 9 (1), 12757 (2019).</li><li>Platzer, 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=Dendritic+cell-bound+IgE+functions+to+restrain+allergic+inflammation+at+mucosal+sites.">Dendritic cell-bound IgE functions to restrain allergic inflammation at mucosal sites.</a> Mucosal Immunology. 8 (3), 516-532 (2015).</li><li>Shinoda, 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=Thy1+IL-7++lymphatic+endothelial+cells+in+iBALT+provide+a+survival+niche+for+memory+T-helper+cells+in+allergic+airway+inflammation.">Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation.</a> Proceedings of the National Academy of Sciences U. S. A. 113 (20), 2842-2851 (2016).</li><li>Nakahashi-Oda, 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=Apoptotic+epithelial+cells+control+the+abundance+of+Treg+cells+at+barrier+surfaces.">Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.</a> Nature Immunology. 17 (4), 441-450 (2016).</li><li>Barrott, J. 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=Modeling+synovial+sarcoma+metastasis+in+the+mouse:+PI3'-lipid+signaling+and+inflammation.">Modeling synovial sarcoma metastasis in the mouse: PI3'-lipid signaling and inflammation.</a> Journal of Experimental Medicine. 213 (13), 2989-3005 (2016).</li><li>Bout&#233;, 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=The+C3HeB/FeJ+mouse+model+recapitulates+the+hallmark+of+bovine+tuberculosis+lung+lesions+following+Mycobacterium+bovis+aerogenous+infection.">The C3HeB/FeJ mouse model recapitulates the hallmark of bovine tuberculosis lung lesions following Mycobacterium bovis aerogenous infection.</a> Veterinary Research. 48 (1), 73 (2017).</li><li>Bal, S. 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=IL-1&#946;,+IL-4+and+IL-12+control+the+fate+of+group+2+innate+lymphoid+cells+in+human+airway+inflammation+in+the+lungs.">IL-1&#946;, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs.</a> Nature Immunology. 17 (6), 636-645 (2016).</li><li>Nakasone, 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=Accumulation+of+gamma/delta+T+cells+in+the+lungs+and+their+roles+in+neutrophil-mediated+host+defense+against+pneumococcal+infection.">Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection.</a> Microbes Infection. 9 (3), 251-258 (2007).</li></ol>

作者没有什么可透露的。

这里描述的方案允许在同一只小鼠中鉴定肺免疫细胞类型和趋化因子或细胞因子的表达。如果需要组织病理学研究，可以在进行单细胞分离步骤之前移除并固定单个叶。这种方法的一个局限性是，如果免疫细胞组成的变化以及趋化因子和/或细胞因子的表达预计在肺的不同叶之间分布不均，则这种方法可能不适用于某些疾病环境。例如，一些细菌，如 结核分枝杆菌， 显示出感染肺的某些叶<...

肺是一个屏障器官，暴露于外部环境，因此不断接受免疫学挑战，如病原体和过敏原1。肺常驻免疫细胞的活化和免疫细胞从外围浸润是清除肺部环境中病原体所必需的。此外，肺常驻免疫细胞保持对共生细菌的耐受性，这表明这些细胞在病原体清除和维持体内平衡1中起作用。肺泡和间质巨噬细胞是肺常驻前哨免疫细胞之一，它们通过模式识别受体感知病原体，并通过吞噬作用2清除这些病原体。肺常驻树突状细胞通过抗原呈递弥合先天性和适应性免疫反应3.此外，激活的局部先天免疫细胞产生细胞因子和趋化因子，这些细胞因子和趋化因子可放大炎症反应并刺激单核细胞、嗜中性粒细胞和淋巴细胞等免疫细胞浸润到肺部1。缺血性卒中已被证明会改变全身免疫力，并导致肺部感染易感性增加。然而，很少有研究评估缺血性卒中后的肺间室，尽管一些研究在炎症条件下检查了它4，5，6，7，8，9。本文描述的方法的目标是同时确定肺病理学、免疫细胞组成以及肺部细胞因子和趋化因子表达水平，以评估肺间室的改变，并评估缺血发作后肺部免疫反应的潜在改变。
这里描述的是用于从小鼠肺部获得单细胞悬浮液以鉴定13种类型的免疫细胞的方案。该方案基于胶原酶D的组织消化，而无需自动组织解离器。此外，我们开发了一种制备组织匀浆的方案，可用于使用基于流式细胞术的多重磁珠阵列确定13种不同细胞因子或趋化因子的表达水平。该方案已成功用于研究缺血性卒中对肺免疫的影响，也可用于其他疾病模型。

<table><tbody><tr><td>B220-APC, clone RA3-6B2</td><td>Biolegend</td><td>103212</td><td>1:200 dilution</td></tr><tr><td>Beadbug 3 position bead homogenizer</td><td>Benchmark Scientific</td><td>D1030</td><td>Tissue homogenizer</td></tr><tr><td>CCR2-BV421, clone SA203G11</td><td>Biolegend</td><td>150605</td><td>1:200 dilution</td></tr><tr><td>CD103-BV421, clone 2E7</td><td>Biolegend</td><td>121422</td><td>1:200 dilution</td></tr><tr><td>CD11b-PE/Cy7, clone M1/70</td><td>Biolegend</td><td>101216</td><td>1:400 dilution</td></tr><tr><td>CD11c-PE/Cy7, clone N418</td><td>Biolegend</td><td>117318</td><td>1:200 dilution</td></tr><tr><td>CD11c-Percp/Cy5.5, clone N418</td><td>Biolegend</td><td>117328</td><td>1:200 dilution</td></tr><tr><td>CD4-BV421, clone GK1.5</td><td>Biolegend</td><td>100443</td><td>1:200 dilution</td></tr><tr><td>CD45-FITC, clone 30-F11</td><td>Biolegend</td><td>103108</td><td>1:200 dilution</td></tr><tr><td>CD64-APC, clone X54-5/7.1</td><td>Biolegend</td><td>139306</td><td>1:200 dilution</td></tr><tr><td>CD8-PE, clone 53-6.7</td><td>Biolegend</td><td>100708</td><td>1:800 dilution</td></tr><tr><td>Collagenase D</td><td>Sigma Aldrich</td><td>11088882001</td><td>Component in the dissociation buffer</td></tr><tr><td>Conical screw cap tube</td><td>ThermoFisher</td><td>02-681-344</td><td>Tube for tissue homogenization</td></tr><tr><td>DNase I</td><td>Sigma Aldrich</td><td>10104159001</td><td>Component in the dissociation buffer</td></tr><tr><td>Fc block CD16/32 antibody</td><td>Biolegend</td><td>101320</td><td>1:100 dilution</td></tr><tr><td>genlteMACS dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>Comparsion of lung digestion with or without mechanical dissociator</td></tr><tr><td>gentleMACS C tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td>Tube for tissue disscoiation with genlteMACS dissociator</td></tr><tr><td>Halt protease and phosphatase inhibitor cocktial</td><td>ThermoFisher</td><td>78442</td><td>Component in the homogenization buffer</td></tr><tr><td>Laser doppler monitor</td><td>Moor</td><td>MOORVMS-LDF</td><td>Blood flow monitoring during tMCAO</td></tr><tr><td>LEGENDplex proinflammatory chemokine panel</td><td>Biolegend</td><td>740451</td><td>Multiplex bead array</td></tr><tr><td>LIVE/DEAD fixable near-IR stain</td><td>ThermoFisher</td><td>L34976</td><td>Use for dead cell exclusion during flow cytometric analysis</td></tr><tr><td>Ly6C-PE, clone HK1.4</td><td>Biolegend</td><td>128008</td><td>1:800 dilution</td></tr><tr><td>Ly6G-BV510, clone 1A8</td><td>Biolegend</td><td>127633</td><td>1:200 dilution</td></tr><tr><td>MCAO suture L56 reusable 6-0 medium</td><td>Doccol</td><td>602356PK10Re</td><td>tMCAO</td></tr><tr><td>MHC II-BV510, clone M5/114.15.2</td><td>Biolegend</td><td>107636</td><td>1:800 dilution</td></tr><tr><td>NK1.1-Percp/Cy5.5, clone PK136</td><td>Biolegend</td><td>108728</td><td>1:200 dilution</td></tr><tr><td>Siglec F-PE, clone E50-2440</td><td>BD Biosciences</td><td>552126</td><td>1:200 dilution</td></tr><tr><td>Silk suture thread, size 6/0</td><td>Fine Science Tools</td><td>18020-60</td><td>tMCAO</td></tr><tr><td>SomnoSuite anesthesia system</td><td>Kent Scientific</td><td>SS-01</td><td>Mouse anaesthetization for tMCAO</td></tr><tr><td>TCRb-BV510, clone H57-897</td><td>Biolegend</td><td>109234</td><td>1:200 dilution</td></tr><tr><td>Zirconia/silica beads, 2.3 mm</td><td>Biospec</td><td>11079125z</td><td>Beads for tissue homogenization</td></tr></tbody></table>

characterization of immune cells and proinflammatory mediators in the pulmonary environment

免疫细胞的扩增、激活和向肺部的运输由多种细胞因子和趋化因子的表达控制，可能因严重的脑损伤而改变。肺炎是缺血性卒中患者死亡的主要原因这一事实证明了这一点。该协议的目的是描述使用多色流式细胞术分析来鉴定小鼠肺部的13种类型的免疫细胞，包括肺泡巨噬细胞，间质巨噬细胞，CD103 +或CD11b +树突状细胞（DC），浆细胞样DC，嗜酸性粒细胞，单核细胞/单核细胞衍生细胞，嗜中性粒细胞，淋巴样来源的T和B细胞，NK细胞和NKT细胞，通过短暂的脑中动脉阻塞诱导缺血性中风。此外，我们使用磁珠匀浆方法描述了肺匀浆的制备，通过多重磁珠阵列结合流式细胞术分析同时确定13种不同细胞因子或趋化因子的表达水平。该方案也可用于研究其他疾病环境中的肺免疫反应，例如传染性肺病或过敏性疾病。

我们最近报道，小鼠缺血性中风诱导改变了肺11的免疫细胞组成。具体而言，短暂性脑缺血增加了肺泡巨噬细胞、嗜中性粒细胞和 CD11b+ DC 的百分比，同时减少了肺区室中 CD4+ T 细胞、CD8+ T 细胞、B 细胞、NK 细胞和嗜酸性粒细胞的百分比。此外，细胞改变对应于肺部多种趋化因子水平的显着降低。这里描述的是一种分离和鉴定肺间室中不同免疫细胞群的方法。这里显示的代表性结?...

Watch this Scientific Journal Video about Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment at JoVE.com

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

该协议描述了使用流式细胞术来鉴定短暂性脑中动脉闭塞后肺部环境中免疫细胞组成，细胞因子谱和趋化因子谱的变化，这是缺血性中风的小鼠模型。

肺环境中免疫细胞和促炎介质的表征

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...

characterization-immune-cells-proinflammatory-mediators-pulmonary

Research

JoVE Journal

Immunology and Infection

3.2K 次观看.  West Virginia University. 该协议描述了使用流式细胞术来鉴定短暂性脑中动脉闭塞后肺部环境中免疫细胞组成，细胞因子谱和趋化因子谱的变化，这是缺血性中风的小鼠模型。

视频: Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

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

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

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

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

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

科研

免疫与感染

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

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 ...

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

在合并臭氧和LPS诱导穆林急性肺损伤期间可视化肺细胞适应

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

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

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

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

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

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

医学

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

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

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

流式细胞术分析鉴定鼠肺先天免疫细胞和适应性免疫细胞

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