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>

<ol>
	<li>Lloyd, C. M., Marsland, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Homeostasis:+Influence+of+Age,+Microbes,+and+the+Immune+System.">Lung Homeostasis: Influence of Age, Microbes, and the Immune System.</a> Immunity. 46 (4), 549-561 (2017).</li><li>Allard, B., Panariti, A., Martin, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+Macrophages+in+the+Resolution+of+Inflammation,+Tissue+Repair,+and+Tolerance+to+Infection.">Alveolar Macrophages in the Resolution of Inflammation, Tissue Repair, and Tolerance to Infection.</a> Frontiers in Immunology. 9, 1777 (2018).</li><li>Hartl, 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=Innate+Immunity+of+the+Lung:+From+Basic+Mechanisms+to+Translational+Medicine.">Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine.</a> Journal of Innate Immunity. 10 (5-6), 487-501 (2018).</li><li>Prass, 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=Stroke-induced+immunodeficiency+promotes+spontaneous+bacterial+infections+and+is+mediated+by+sympathetic+activation+reversal+by+poststroke+T+helper+cell+type+1-like+immunostimulation.">Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation.</a> Journal of Experimental Medicine. 198 (5), 725-736 (2003).</li><li>Smith, C. 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=Interleukin-1+receptor+antagonist+reverses+stroke-associated+peripheral+immune+suppression.">Interleukin-1 receptor antagonist reverses stroke-associated peripheral immune suppression.</a> Cytokine. 58 (3), 384-389 (2012).</li><li>McCulloch, L., Smith, C. J., McColl, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic-mediated+loss+of+splenic+marginal+zone+B+cells+contributes+to+infection+susceptibility+after+stroke.">Adrenergic-mediated loss of splenic marginal zone B cells contributes to infection susceptibility after stroke.</a> Nature Communications. 8 (1), 15051 (2017).</li><li>Dames, 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=Immunomodulatory+treatment+with+systemic+GM-CSF+augments+pulmonary+immune+responses+and+improves+neurological+outcome+after+experimental+stroke.">Immunomodulatory treatment with systemic GM-CSF augments pulmonary immune responses and improves neurological outcome after experimental stroke.</a> Journal of Neuroimmunology. 321, 144-149 (2018).</li><li>Jin, R., Liu, S., Wang, M., Zhong, W., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+CD147+Attenuates+Stroke-Associated+Pneumonia+Through+Modulating+Lung+Immune+Response+in+Mice.">Inhibition of CD147 Attenuates Stroke-Associated Pneumonia Through Modulating Lung Immune Response in Mice.</a> Frontiers in Neurology. 10, 853 (2019).</li><li>Yu, Y. R., 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+Protocol+for+the+Comprehensive+Flow+Cytometric+Analysis+of+Immune+Cells+in+Normal+and+Inflamed+Murine+Non-Lymphoid+Tissues.">A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.</a> PLoS One. 11 (3), 0150606 (2016).</li><li>Rousselet, E., Kriz, J., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+intraluminal+MCAO:+cerebral+infarct+evaluation+by+cresyl+violet+staining.">Mouse model of intraluminal MCAO: cerebral infarct evaluation by cresyl violet staining.</a> Journal of Visualized Experiments. (69), e4038 (2012).</li><li>Farris, B. Y., 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=Ischemic+stroke+alters+immune+cell+niche+and+chemokine+profile+in+mice+independent+of+spontaneous+bacterial+infection.">Ischemic stroke alters immune cell niche and chemokine profile in mice independent of spontaneous bacterial infection.</a> Immunity, Inflammation and Diseases. 7 (4), 326-341 (2019).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Lehmann, J. 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的倾向21。在这种情况下，可能需要在波瓣之间进行比较。
从肺部分离单细胞方案的浓度，孵育时间和温度严重影响免疫细胞从肺部的恢复。胶原酶 D 的质量对于获得最佳结果至关重要，如果使用胶原酶 D 的不同来源，则应进行测试。组织的过度消化导致细胞死亡的增加;然而，组织的消化不足导致免疫细胞的产量低，尤其是巨噬细胞和DC。
我们使用3种抗体组合来确定肺部的13个免疫细胞群，每组含有5-7个抗体和一个LIVE/DEAD染色。如果从样品中获得的细胞数量有限，则可以组合每组中的抗体。 然而，当抗体数量增加时出现的一个主要问题是，流式细胞仪上荧光信号的补偿可能具有挑战性，特别是在炎症条件下将细胞与髓系细胞区分开来时。额外的抗体混合物可用于鉴定单细胞悬浮液中的其他先天免疫细胞，例如先天淋巴细胞和γδ T细胞，这些细胞有助于肺部的免疫反应22，23。由于多路复用阵列取肺的一个肺叶，因此无法确定和比较肺中每种细胞类型的确切数量，这构成了该方法的限制。为了解决这个问题，可以从假小鼠和TMCAO诱导的小鼠的肺部分离出一定数量的细胞。在这种情况下，可以准确地比较两组之间每种免疫细胞类型的绝对数量。此外，这种方法不允许确定肺中免疫细胞的定位。这可以通过对肺切片进行免疫组织化学检查来实现。
综上所述，该方案用于研究缺血性卒中对肺免疫的影响，但也可用于研究其他疾病模型，如感染和过敏。

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 border="0" cellpadding="0" cellspacing="0" style="width:932px;" width="931">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">B220-APC, clone RA3-6B2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103212</td>
			<td style="width:412px;">1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Beadbug 3 position bead homogenizer</td>
			<td style="width:129px;">Benchmark Scientific</td>
			<td style="width:101px;">D1030</td>
			<td>Tissue homogenizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CCR2-BV421, clone SA203G11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">150605</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD103-BV421, clone 2E7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">121422</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11b-PE/Cy7, clone M1/70</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101216</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-PE/Cy7, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117318</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD11c-Percp/Cy5.5, clone N418</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">117328</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD4-BV421, clone GK1.5</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100443</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD45-FITC, clone 30-F11</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">103108</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD64-APC, clone X54-5/7.1</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">139306</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">CD8-PE, clone 53-6.7</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">100708</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Collagenase D</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>11088882001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Conical screw cap tube</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">02-681-344</td>
			<td>Tube for tissue homogenization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">DNase I</td>
			<td style="width:129px;">Sigma Aldrich</td>
			<td>10104159001</td>
			<td>Component in the dissociation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Fc block CD16/32 antibody</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">genlteMACS dissociator</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Comparsion of lung digestion with or without mechanical dissociator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">gentleMACS C tubes</td>
			<td style="width:129px;">Miltenyi Biotec</td>
			<td style="width:101px;">130-093-237</td>
			<td>Tube for tissue disscoiation with genlteMACS dissociator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Halt protease and phosphatase inhibitor cocktial</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">78442</td>
			<td>Component in the homogenization buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">Laser doppler monitor</td>
			<td style="width:129px;">Moor</td>
			<td style="width:101px;">MOORVMS-LDF</td>
			<td>Blood flow monitoring during tMCAO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">LEGENDplex proinflammatory chemokine panel</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">740451</td>
			<td>Multiplex bead array</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">LIVE/DEAD fixable near-IR stain</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:101px;">L34976</td>
			<td>Use for dead cell exclusion during flow cytometric analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6C-PE, clone HK1.4</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">128008</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Ly6G-BV510, clone 1A8</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">127633</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:289px;">MCAO suture L56 reusable 6-0 medium</td>
			<td style="width:129px;">Doccol</td>
			<td style="width:101px;">602356PK10Re</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">MHC II-BV510, clone M5/114.15.2</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">107636</td>
			<td>1:800 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">NK1.1-Percp/Cy5.5, clone PK136</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">108728</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Siglec F-PE, clone E50-2440</td>
			<td style="width:129px;">BD Biosciences</td>
			<td style="width:101px;">552126</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Silk suture thread, size 6/0</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td style="width:101px;">18020-60</td>
			<td>tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">SomnoSuite anesthesia system</td>
			<td style="width:129px;">Kent Scientific</td>
			<td style="width:101px;">SS-01</td>
			<td>Mouse anaesthetization for tMCAO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">TCRb-BV510, clone H57-897</td>
			<td style="width:129px;">Biolegend</td>
			<td style="width:101px;">109234</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">Zirconia/silica beads, 2.3 mm</td>
			<td style="width:129px;">Biospec</td>
			<td style="width:101px;">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果来自经过TMCAO诱导和假手术的小鼠。
我们使用3组抗体组合鉴定出肺部13个不同的免疫细胞群（L1-L13），每组含有5-7个抗体（图1）。在每组中使用活/死染色剂排除死细胞。用于区分不同免疫细胞类型的抗体和标志物列于 表1中。在第1组中鉴定出肺泡和间质巨噬细胞，CD103 + DC，CD11b + DC和嗜酸性粒细胞（图1A，B）。在第2组中鉴定出促炎单核细胞和嗜中性粒细胞（图1C）。在炎症反应期间，单核细胞迁移到炎症部位，在那里这些细胞分化成单核细胞衍生的抗原呈递细胞（mo-APC）14。Ly6C和CCR2的下调是单核细胞分化的特征，可以使用Set 215进行评估。 使用组3鉴定CD4 + T细胞，CD8 + T细胞，B细胞，浆细胞DC，NK细胞和NKT细胞（图1D）。
为了确定我们的单细胞分离方案的质量，我们将使用手动方法分离的活细胞和CD45 +免疫细胞的数量与使用市售组织解离器分离的细胞数量进行了比较（参见材料表），该分离器通常用于从组织中分离细胞16，17，18，19，20.在后一种方案中，在注射解离缓冲液后，肺叶被转移到解离器特异性管中（参见材料表），并且使用37C_m_LDK_1程序消化组织。两种方法之间的活细胞总数，CD45 +细胞的百分比和CD45 +细胞的总数具有可比性（图2A-C）。使用两种方案的CD45 +细胞中的细胞死亡百分比约为10%（图2D）。这些结果表明，这里提出的方案允许在没有自动组织解离器的帮助下以高产量和质量恢复细胞。
使用市售的多重测定与流式细胞术分析相结合，使用25μL样品测定13种趋化因子的浓度（图3）。FSC/SSC首先鉴定出两种不同尺寸的磁珠（图3A）。每个磁珠都涂有6-7个一抗，这可以通过APC通道中的荧光强度来区分（图3B）。样品中的趋化因子水平与PE通道中的荧光强度成正比，这可以通过MFI确定（图3C）。通过将每个趋化因子的MFI值与用已知浓度的趋化因子构建的标准曲线进行比较，可以确定样品中的浓度（每mg组织）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61359/61359fig1.jpg"> 图1:缺血性中风后使用胶原酶D消化后从肺部鉴定出13种免疫细胞类型。 在TMCAO或假手术后24小时切除肺组织，通过流式细胞术分析肺部的免疫细胞，由表1中列出的表面标记物定义。（A）在抗体组1中，首先在西格莱克F和CD11b上对CD45+活细胞（*）进行门控，以鉴定表达CD11c和MHC II的肺泡巨噬细胞（L1）和不表达CD11c和MHC II的嗜酸性粒细胞（L2）。（B）然后将A中的西格莱克F-群体（**）中的细胞门控以确定CD103和CD11b的表达。进一步对细胞进行门控以确定CD11c和MHC II的表达。CD103+ DC 同时表达 CD11c 和 MHC II （L3），但不表达 CD64。CD11b hi群体（****）进一步被门控以确定CD64和MHC II的表达。CD11b+ DC 表达 MHC II，但不表达 CD64 （L4），而间质巨噬细胞 （L5） 同时表达这两种标记。（C）在抗体组2中，对A中的CD45+活细胞进行门控以确定CD11b和Ly6C的表达。Ly6C高细胞（*）代表维持高水平CCR2表达的未分化单核细胞（L6，中间图），而Ly6C低细胞（**）包含Ly6G-（L6，右图）和Ly6G +嗜中性粒细胞（L7）的分化单核细胞的混合群体。（D）在抗体组3中，对A中的CD45 +活细胞进行门控以确定CD11c和B220的表达，以鉴定B细胞（L8）和浆细胞DC（L9）。然后将CD11c-和B220-群体（*）进行门控以确定CD4和CD8的表达，以鉴定CD4 + T细胞（L10）和CD8 + T细胞（L11）。进一步门控CD4-CD8-群体（**）以确定NK1.1和TCRb的表达，以鉴定NK细胞（L12）和NKT细胞（L13）。图中显示了12只C57BL / 6J小鼠在TMCAO和假手术后的代表性图。该图的部分内容经许可转载自先前发表的文献11 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61359/61359fig1large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61359/61359fig2.jpg"> 图2:手动解离方法与使用 组织解离器从肺部分离单个细胞 之间的比较。（自动对照）比较细胞总数，CD45 +细胞的百分比和CD45 +细胞的总数。图中显示了3个独立实验的综合结果。NS:无统计学意义。（D）代表性图，以确定分离后死亡CD45 +细胞的百分比。图中显示了来自3个独立实验的代表性图。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61359/61359fig2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61359/61359fig3.jpg"> 图3:缺血性中风抑制肺部多种趋化因子的产生。 （自动对照）代表性图显示通过多重磁珠阵列测定肺部13种趋化因子的水平。 （A）FSC/SSC门用于识别不同尺寸的磁珠A和B。（B）包被在磁珠上的一抗可以通过APC通道中的荧光强度来区分。（C）样品中的趋化因子水平与PE通道中的荧光强度成正比。图中显示了假手术后12只C57BL / 6J小鼠的代表性图。（D）肺组织在TMCAO或假手术后24小时均质化。单个动物肺部13个趋化因子的水平通过多重磁珠阵列确定。显示的数据是三个独立实验的综合结果，每组n = 11-12只动物。*， P < 0.05;**， P < 0.01;，P < 0.001。NS，在统计上没有区别。该图的部分内容经许可转载自先前发表的文献11 。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61359/61359fig3large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:keep-together.within-page="1"><tbody><tr><td>抗体</td>
			<td>克隆</td>
			<td>免疫细胞类型</td>
			<td>人口</td>
			<td>表面标记表达式</td>
		</tr><tr><td>CD45-菲特</td>
			<td>30-F11</td>
			<td>阿尔维洛巨噬细胞</td>
			<td>L1</td>
			<td>CD45+ 西格莱克 F+ CD11b-</td>
		</tr><tr><td>西格莱克 F-聚乙烯</td>
			<td>E50-2440型</td>
			<td>嗜酸性粒细胞</td>
			<td>二层</td>
			<td>CD45+ 西格莱克 F+ CD11b+</td>
		</tr><tr><td>CD11c-珍珠/细胞5.5</td>
			<td>N417</td>
			<td>CD103+ 直流电</td>
			<td>低三层</td>
			<td>CD45+ 西格莱克 F- CD11b- CD103+ CD11c+ 兆格力 II+</td>
		</tr><tr><td>CD11b-聚乙烯/环氧乙烷7</td>
			<td>M1/70</td>
			<td>CD11b+ 直流电</td>
			<td>四层</td>
			<td>CD45+ 西格莱克 F- CD11b 嗨 CD103- CD64- 兆赫二世+</td>
		</tr><tr><td>CD64-断续器</td>
			<td>X54-5/7.1</td>
			<td>间质巨噬细胞</td>
			<td>L5</td>
			<td>CD45+ 西格莱克 F- CD11b 嗨 CD103- CD64+ 兆目二世+</td>
		</tr><tr><td>CD103-BV421</td>
			<td>2E7</td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>二型-BV510型</td>
			<td>M5/114.15.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>活/死-APC/Cy7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>CD45-菲特</td>
			<td>30-F11</td>
			<td>单核细胞/模数转换器</td>
			<td>低8层</td>
			<td>CD45+ CD11b 嗨 Ly6C hi/int CCR2+/- Ly6G-</td>
		</tr><tr><td>聚醚醚酮羧酸</td>
			<td>港币1.4元</td>
			<td>中性 粒 细胞</td>
			<td>L9</td>
			<td>CD45+ CD11b 嗨 Ly6C int CCR2- Ly6G+</td>
		</tr><tr><td>CD11b-聚乙烯/环氧乙烷7</td>
			<td>M1/70</td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>CCR2-BV421</td>
			<td>南非203G11</td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>利6G-BV510</td>
			<td>1A8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>活/死-APC/Cy7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>CD45-菲特</td>
			<td>30-F11</td>
			<td>等离子体 DC</td>
			<td>L6</td>
			<td>CD45+ B220+ CD11c+</td>
		</tr><tr><td>镉8-聚乙烯</td>
			<td>53-6.7</td>
			<td>B细胞</td>
			<td>L7</td>
			<td>CD45+ B220+ CD11c-</td>
		</tr><tr><td>NK1.1-珍珠/氰基5.5</td>
			<td>PK136</td>
			<td>CD4+ T 细胞</td>
			<td>L10</td>
			<td>CD45+ B220- CD11c- CD4+ CD8-</td>
		</tr><tr><td>CD11c-聚乙烯/环氧乙烷7</td>
			<td>N417</td>
			<td>CD8+ T 细胞</td>
			<td>L11</td>
			<td>CD45+ B220- CD11c- CD4- CD8+</td>
		</tr><tr><td>断续器-B220</td>
			<td>抗坏反应组 3-6B2</td>
			<td>核糖核酸细胞</td>
			<td>L12</td>
			<td>CD45+ B220- CD11c- CD4- CD8- NK1.1+ TCRb-</td>
		</tr><tr><td>CD4-BV421</td>
			<td>吉克1.5</td>
			<td>耐克特电池</td>
			<td>L13</td>
			<td>CD45+ B220- CD11c- CD4- CD8- NK1.1+ TCRb+</td>
		</tr><tr><td>断续器-BV510</td>
			<td>H57-596</td>
			<td></td>
			<td></td>
			<td></td>
		</tr><tr><td>活/死-APC/Cy7</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr></tbody></table>表1: 表面标志物和抗体组合，用于确定TMCAO后从肺部分离的免疫细胞。

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

Nanyang Technological University

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

右心室收缩压测量组合在小鼠的肺和免疫组织样品的收获

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪组织免疫细胞的分离

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...