我们感谢杰夫·塔克，埃丽卡·斯卡皮尼，和阿格奈什·贾诺沙齐他们用显微镜的帮助下，利贡·佩罗她的鼠标殖民地的管理，朱·切和迈克尔·山德森与组织切片机的帮助下，和迈克尔·费斯勒和德里克该隐的批判性阅读手稿。这项工作是由NIEHS，美国国立卫生研究院（ZIA ES102025-09），后者又由卫生和人类服务部赞助的校内分行资助。

本文中所描述的动物实验过程由NIEHS动物护理和使用委员会（IACUC）的批准。 1.肺制备<ol><li> 6和12周龄无特定病原体条件之间家鼠按照机构动物护理和使用委员会提供的指导原则。 注:成像小鼠可以是天真的，或经处理的，这取决于每个研究人员的具体利益。在这里，我们描述了在天真的小鼠和在用100μg卵清蛋白（OVA）和0.1微克的脂多糖（LPS）处理的小鼠中的免疫细胞定位，使用磷酸盐缓冲盐水（PBS）在50微升的总体积的车辆。到oropharyngeally灌输OVA / LPS进入气道，将动物用异氟烷麻醉，吸入并通过用橡胶带齿垂直悬挂。舌头轻轻地用钳子抓住并持有偏向一侧，防止吞咽和50＆＃181;保藏在口腔后部的OVA / LPS溶液的L，以先前描述的17。 </li><li>安乐死用戊巴比妥钠（100毫克/千克）的腹膜内注射小鼠，并钉住动物对于聚苯乙烯基。 </li><li>通过从腹部到颚的中间切开皮肤和腹膜打开用剪刀腹腔。用钳子或剪刀的钝边一边拉肠，和剪断下腔静脉从肺部排出血液程。穿刺用剪刀的尖端隔膜以允许所述左肋膨胀，小心不要切断肺部。 </li><li>清除唾液腺和通过抓住组织并手动从与钳子底层气管拉动远离其它组织从气管程。使使用细镊子或剪刀软骨厚带的前侧气管一小切口，小心不要一路砍虽然TRA谢。该切口将只是大到足以让一个20号针通过。 </li><li>滑动的1.5英寸，20号针上的聚乙烯管的截面，留下约1cm管超过针的端部。切管以大约45°的角度通过拉伸0.8毫升热水（40℃）2％低熔点琼脂糖向上穿过针斜面端部，附接针1mL注射器，并加载注射器。 </li><li>将管的一端插入气管的切口，并缓缓注入琼脂糖进入肺部。而无需移动针或注射器，用胶带将注射器的聚苯乙烯基础，以确保针头留在气管中。 注意:注射后，肺部将胸腔内较大和充气。右叶首先展开，然后左叶。如果只有一个叶扩大，针已经插入过深（过去支气管），而且有必要在继续之前把它拉出来咯。的这部分过程需要相对较快地完成，琼脂糖开始凝固之前。 </li><li>将鼠标，与基座和在气管仍插注射器，在冷室或冰箱中至少10分钟。如果有必要，动物可以在那里保存长达几个小时，即使通过视频显微镜在进行图像细胞迁移。 </li><li>一旦鼠标是冷的，用剪刀仔细切除琼脂糖充气肺，并将它们放置在一个3 cm培养皿用冰冷的PBS。在冰上保持。 </li><li>选择用于组织切片所需的波瓣（ 例如 ，右上叶）。确保肺（它连接到气管）的内饰部分是面朝下的菜。 注:所用的叶和活塞上的取向将决定血管和气管，这将是可见的大小。这里所描述的方位非常适合大型航空公司的可视化和实质。老鼠有一个大的左叶和右四个小瓣。对于以下介绍过敏原成通过口咽或鼻内抽吸肺PCLS成像，右上叶通常剖开，部分是因为它是一种方便的大小，而且还因为吸入剂在其内均匀地分散。然而，其他叶，尤其是左叶，也可以研究。当比较小鼠品系或治疗，它可以比较同叶是很重要的。 </li></ol> 2.肺切片注意:请用自动切片机，金属冷却块，柱塞和注射器金属片，根据制造商的说明。 <ol><li>放的所有目的的小滴，没有润凝胶强力胶到柱塞和散布胶水的圆周运动，同时注意不要让胶水接触金属注射器的侧面。如果胶水接触到注射器的两侧，将胶针筒和柱塞在一起。 </li><li>覆盖用胶水活塞后，立即轻轻抓住用与气管侧朝下钳子切叶，DAB它上的组织，以除去过量的液体，然后小心地将其放置在柱塞的顶部。剪掉延伸超出所述柱塞的边缘的任何额外的组织。 </li><li>向下移动柱塞，使得金属注射器的侧面向上移动，并通过组织，从而形成井在底部胶合，不超过几个厘米深肺。周围的金属注射器的底部胶带举行的地方这个位置。 </li><li>小心倾2％低熔点琼脂糖在40℃下到井中以便它只是覆盖波瓣的顶部。 </li><li>包围与冰冷的冷却块中的金属注射器和冷却叶和琼脂糖为1 - 2分钟。 注:周围组织中的琼脂糖的更短的时间冷却可以切片，这可能会导致切割不均匀PCLS期间导致琼脂糖的崩解。 </li><li>加载样品注射器到自动限幅器和填充用冰冷的PBS缓冲罐。对齐<html>步骤与试样注射器马达驱动，并从底部移除胶带片。转切换到快进（FF）的位置，直到所述步进电机驱动器刚好接触到柱塞的背面。 </li><li>对齐与样品注射器新的刀片。集合组织厚度，连续/单切片，振荡和速度在限幅器。例如，组织厚度:150微米，连续切削（续），振荡:9，和速度:3 - 4.按开始。通过以上的设置需要调整到特定的自动切片机规格。 </li><li>使用薄的抹刀或画笔，收集PCLS一次一个，因为它们落入缓冲罐和将它们放置在含有冰冷的PBS 24孔板中。 注:保持片，以保持实验之间的一致性是非常重要的。虽然每个肺是根据年龄，性别和重量不同，则10 个切片，例如，通常能产生类似大小的气道。 </li></ol>ove_title"&gt; 3。抗体染色<ol><li>设计基于对感兴趣的细胞类型的荧光标记抗体的面板中，考虑到潜在的荧光光谱重叠和显微镜的检测能力。 注意:作为一个例子，对于DC子集检测一个面板是如下:的CD11c /α-X整 - BrilliantViolet（BV）605（激发:405纳米，峰值发射:605纳米），CD324 / E-钙粘蛋白 - 的Alexa 488（AF488 ）（激发:488纳米，峰值发射:519纳米），CD88 / C5Ra1 - 藻红蛋白（PE）（激发:561纳米，峰值发射:578纳米），CD103 /α-ë整 - 别藻蓝蛋白（APC）（激发:633纳米，峰值发射:660纳米）。荧光光谱查看器工具（参阅材料清单）是设计的面板非常有用。 </li><li>使含有所需抗体的抗体混合物。 <ol><li>对于静态PCLS成像，添加800μL的染色缓冲液，100微升弗克·布洛克，50μL正常小鼠血清，和50μL正常大鼠血清到1.5mL microcentrifugation管1毫升的总体积。活细胞成像，使用含有10％FBS（的Leibovitz-10）代替染色缓冲液800μL的Leibovitz培养基（1×）。 </li><li>添加抗体来调节每种抗体的所期望的终浓度。转移抗体溶液到3cm的盘，并保持在黑暗中待用。 注:（ - 5微克/毫升通常1种）的最终浓度需要为每个单独的抗体进行优化。 </li></ol></li><li>选择与所述兴趣，如大气道或肺的周围的解剖区域中的PCLS，并在含有1种毫升抗体溶液中的3-cm培养皿放置。 </li><li>污点PCLS在黑暗中，在冰上，慢慢摇动30或60分钟。 </li><li>对于静态PCLS成像，进行到该部分4。对于活细胞成像，则继续执行第5节。 </li></ol> 4. PCLS的静态影像<ol><li>后用抗体PCLS的60分钟温育后，除去从培养皿抗体溶液和冲洗吨他用1个毫升冰冷的PBS切片两次。 </li><li>移液管加入50μLPBS涂到3厘米的圆形玻璃底培养皿中。使用刮刀或画笔，染色PCLS转移到滴，轻轻操纵切片，直到它是平坦的并且展开。用吸管取出PBS。切片应位于尽可能平坦。上述程序需要快速执行，以避免光漂白。 </li><li>放置室温下的安装介质1滴置于该片中，轻轻地滴在玻璃盖玻片到板的孔中。保持PCLS在黑暗中在4℃下最多成像前一个小时。 </li><li>当共焦显微镜下观察，使用20X物镜（参见材料列表 ）。使用"瓦片"工具中的"凸包"的设置来定位和标记组织的边缘。这将有助于减少瓷砖扫描的时间。 </li><li>而设置的z堆栈范围，验证在切片的多个区域，特别是沿着边缘，该组范围是合适的。 注:Z-范围将可能需要比组织本身的厚度更大，因为它是很难得到的组织奠定完全平坦。例如，具有150微米厚的PCLS中，z堆栈范围将可能需要被设置为200 - 250微米，以适应切片凸点和曲线。取决于Z-堆叠范围和组织的尺寸，每个全肺扫描将需要7之间 - 12小时用20X物镜。每个肺是唯一的，并且设置必须进行调整，每个实验。 </li></ol> PCLS 5.活细胞成像<ol><li> PCLS的30分钟温育的抗体后，从培养皿的抗体溶液中，用1毫升冷的Leibovitz-10冲洗切片两次。 </li><li>移液管50的新鲜的LeibovitzμL-10成一个一个腔玻璃罩（8个时隙）的时隙。用铲子将PCLS转印至介质，轻轻地操纵它，直到片是扁平的而展开。上述程序需要迅速执行，以避免光漂白。 </li><li>除去使用移液管的50μL的平台。切片要被躺在尽可能平坦。 注意:如果切片的边缘抵靠腔室的壁垂直向上翻转这是可接受的。此外，如果可用的话，金属铂的重量可用于在进行下一步骤之前，以锚定条带。见所附的材料清单为铂线的一个例子，可以被切割和弯曲成小的权重。 </li><li>在4℃冷室中，混合100微升生长因子减少，用100μL的Leibovitz-10不含酚红的细胞外基质的。使用预冷的吸头用于此，或细胞外基质将在尖端凝固。 注:基质和介质的这一比率（1:1）产生足够的致密保持PCLS下在组织培养条件的凝胶。 </li><li>轻轻吸取基质混合，并认真吸取在PCLS的顶部，确保凝胶那张露顶纳克片，并且所述片不会在凝胶的顶部上浮。凝胶应该工作作为权重，下锚定PCLS到板的底部。 </li><li>小心地将腔玻璃罩转移到37℃培养箱并让基质固化〜5分钟。 </li><li>传送整个腔室盖玻片到共焦显微镜的预热阶段。保持在整个成像37℃的室中。使用Leibovitz的介质时，不将细胞培养过程中所需的CO 2的供应。 </li><li>设定基于帧之间的所需间隔的z堆栈范围和瓦范围内。当共焦显微镜下观察，使用20X物镜（参见材料列表）。使用"瓦片"工具来表示的在组织中感兴趣的区域，使用"中心网格"设置（ 例如 ，2×2网格为中心的）。 </li><li>而设置的z堆栈范围，验证在切片所设置的范围是适当的多个区域。 注:ZR安格将可能等于组织的厚度。每个肺是唯一的，并且设置必须进行调整，每个实验。 2×2的瓷砖和10的z堆叠通常将导致〜1帧/ 2分钟。确保自动对焦功能开始在实验过程中成像期间以最小化的xy和z漂移之前被激活。 </li></ol>

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	<li>Schneider, T., van Velzen, D., Moqbel, R., Issekutz, 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=Kinetics+and+quantitation+of+eosinophil+and+neutrophil+recruitment+to+allergic+lung+inflammation+in+a+brown+Norway+rat+model.">Kinetics and quantitation of eosinophil and neutrophil recruitment to allergic lung inflammation in a brown Norway rat model.</a> Am J Respir Cell Mol Biol. 17 (6), 702-712 (1997).</li><li>Vermaelen, K. Y., Carro-Muino, I., Lambrecht, B. N., Pauwels, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+migratory+dendritic+cells+rapidly+transport+antigen+from+the+airways+to+the+thoracic+lymph+nodes.">Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.</a> J Exp Med. 193 (1), 51-60 (2001).</li><li>Sung, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+lung+CD103+(alphaE)-beta7+integrin-positive+epithelial+dendritic+cell+population+expressing+Langerin+and+tight+junction+proteins.">A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.</a> J Immunol. 176 (4), 2161-2172 (2006).</li><li>Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lasker+Basic+Medical+Research+Award.+Dendritic+cells:+versatile+controllers+of+the+immune+system.">Lasker Basic Medical Research Award. Dendritic cells: versatile controllers of the immune system.</a> Nat Med. 13 (10), 1155-1159 (2007).</li><li>Jakubzick, 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=Lymph-migrating,+tissue-derived+dendritic+cells+are+minor+constituents+within+steady-state+lymph+nodes.">Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes.</a> J Exp Med. 205 (12), 2839-2850 (2008).</li><li>Julia, V., 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+restricted+subset+of+dendritic+cells+captures+airborne+antigens+and+remains+able+to+activate+specific+T+cells+long+after+antigen+exposure.">A restricted subset of dendritic cells captures airborne antigens and remains able to activate specific T cells long after antigen exposure.</a> Immunity. 16 (2), 271-283 (2002).</li><li>Nakano, 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=Migratory+properties+of+pulmonary+dendritic+cells+are+determined+by+their+developmental+lineage.">Migratory properties of pulmonary dendritic cells are determined by their developmental lineage.</a> Mucosal Immunol. 6 (4), 678-691 (2013).</li><li>Nakano, 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=Complement+receptor+C5aR1/CD88+and+dipeptidyl+peptidase-4/CD26+define+distinct+hematopoietic+lineages+of+dendritic+cells.">Complement receptor C5aR1/CD88 and dipeptidyl peptidase-4/CD26 define distinct hematopoietic lineages of dendritic cells.</a> J Immunol. 194 (8), 3808-3819 (2015).</li><li>Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+lung+physiology+in+health+and+disease+with+lung+slices.">Exploring lung physiology in health and disease with lung slices.</a> Pulm Pharmacol Ther. 24 (5), 452-465 (2011).</li><li>Liberati, T. A., Randle, M. R., Toth, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+lung+slices:+a+powerful+approach+for+assessment+of+lung+pathophysiology.">In vitro lung slices: a powerful approach for assessment of lung pathophysiology.</a> Expert Rev Mol Diagn. 10 (4), 501-508 (2010).</li><li>Parrish, A. R., Gandolfi, A. J., Brendel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+tissue+slices:+applications+in+pharmacology+and+toxicology.">Precision-cut tissue slices: applications in pharmacology and toxicology.</a> Life Sci. 57 (21), 1887-1901 (1995).</li><li>Bergner, A., Sanderson, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+stimulates+Ca2++oscillations+and+contraction+in+airway+smooth+muscle+cells+of+mouse+lung+slices.">ATP stimulates Ca2+ oscillations and contraction in airway smooth muscle cells of mouse lung slices.</a> Am J Physiol Lung Cell Mol Physiol. 283 (6), L1271-L1279 (2002).</li><li>Martin, C., Uhlig, S., Ullrich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Videomicroscopy+of+methacholine-induced+contraction+of+individual+airways+in+precision-cut+lung+slices.">Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices.</a> Eur Respir J. 9 (12), 2479-2487 (1996).</li><li>Henjakovic, 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=Ex+vivo+testing+of+immune+responses+in+precision-cut+lung+slices.">Ex vivo testing of immune responses in precision-cut lung slices.</a> Toxicol Appl Pharmacol. 231 (1), 68-76 (2008).</li><li>Henjakovic, 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=Ex+vivo+lung+function+measurements+in+precision-cut+lung+slices+(PCLS)+from+chemical+allergen-sensitized+mice+represent+a+suitable+alternative+to+in+vivo+studies.">Ex vivo lung function measurements in precision-cut lung slices (PCLS) from chemical allergen-sensitized mice represent a suitable alternative to in vivo studies.</a> Toxicol Sci. 106 (2), 444-453 (2008).</li><li>Paddenberg, 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=Hypoxic+vasoconstriction+of+partial+muscular+intra-acinar+pulmonary+arteries+in+murine+precision+cut+lung+slices.">Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices.</a> Respir Res. 7, 93 (2006).</li><li>Wilson, R. 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=Allergic+sensitization+through+the+airway+primes+Th17-dependent+neutrophilia+and+airway+hyperresponsiveness.">Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness.</a> Am J Respir Crit Care Med. 180 (8), 720-730 (2009).</li><li>Schlitzer, 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=Identification+of+cDC1-+and+cDC2-committed+DC+progenitors+reveals+early+lineage+priming+at+the+common+DC+progenitor+stage+in+the+bone+marrow.">Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.</a> Nat Immunol. 16 (7), 718-728 (2015).</li><li>Baluk, P., 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=Preferential+lymphatic+growth+in+bronchus-associated+lymphoid+tissue+in+sustained+lung+inflammation.">Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.</a> Am J Pathol. 184 (5), 1577-1592 (2014).</li><li>Jurisic, G., Iolyeva, M., Proulx, S. T., Halin, C., Detmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+cell+antigen+1+(Thy1,+CD90)+is+expressed+by+lymphatic+vessels+and+mediates+cell+adhesion+to+lymphatic+endothelium.">Thymus cell antigen 1 (Thy1, CD90) is expressed by lymphatic vessels and mediates cell adhesion to lymphatic endothelium.</a> Exp Cell Res. 316 (17), 2982-2992 (2010).</li><li>Truman, L. 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=ProxTom+lymphatic+vessel+reporter+mice+reveal+Prox1+expression+in+the+adrenal+medulla,+megakaryocytes,+and+platelets.">ProxTom lymphatic vessel reporter mice reveal Prox1 expression in the adrenal medulla, megakaryocytes, and platelets.</a> Am J Pathol. 180 (4), 1715-1725 (2012).</li><li>Wigle, J. T., Oliver, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prox1+function+is+required+for+the+development+of+the+murine+lymphatic+system.">Prox1 function is required for the development of the murine lymphatic system.</a> Cell. 98 (6), 769-778 (1999).</li></ol>

作者有没有利益冲突申报。

这里描述的协议最初开发可视化肺内疾病预防控制中心两个子集的位置。然而，该协议可以很容易地适合于研究许多不同细胞类型，同时保持细胞活力和肺的三维结构。后者的特征是一个重要的优点在细胞培养系统，并促进稀有细胞类型的标识。该方法依赖于从肺PCLS的产生，和抗体的适当组合，以确定特定的细胞类型，同时最小化背景染色。在很大程度上，这是一个经验的运动，因为，在其他应...

以下促炎性刺激物的吸入，如脂多糖（LPS），有免疫细胞的协调运动成，内，并从肺。例如，嗜中性粒细胞迅速募集至肺实质及气道。此外，被称为常规树突细胞（cDC上）的一些专门的抗原呈递细胞进行相对复杂的迁移模式1，2。 cDC上可以使用流式细胞术来鉴定，部分地基于它们的表面标记的显示，CD11c的上。 DC的不同子集可以通过CD103和CD11b 3的差动表面表达来区分。当获取吸入抗原，一些cDC上退出肺并通过淋巴管迁移到肺引流淋巴结（LN中），其中它们呈现的肽抗原特异性T细胞4。这是在适应性免疫R的起始的关键早期事件esponses。不知什么原因，然而，并非收购吸入抗原所有cDC上离开肺，和许多这些细胞保持在该机构数月5，6。该观察可以通过将这些细胞的发育祖先可以部分地解释，因为单核细胞衍生的CD11c +缺乏趋化因子受体，CCR7的细胞，无法迁移到区域性淋巴结7,8。这很可能是疾病预防控制中心迁移潜力还确定，至少部分地由肺内的解剖位置。然而，在肺cDC上的这些不同的群体的精确定位不是完全表征。免疫细胞定位的肺中，并引导其分子的一种改进的知识，是需要更好地了解肺的免疫系统变得如何激活。 PCLS正在增加LY用来作为显现细胞的定位和细胞-细胞相互作用的离体方法中，同时维持肺结构9，10的结构完整性。 PCLS已用于研究许多物种，包括老鼠，牛，猴，羊，马和人11的肺。该技术的主要优点是，大约20片可以从小鼠肺的单个叶来制备，由此减少所需要的个体实验动物的数量。实际上，所有的免疫细胞类型（包括DC），巨噬细胞，中性粒细胞和T细胞，存在于PCLS和维持其正常结构。 PCLS也可以用来研究钙信号传导与气道和平滑肌细胞收缩与乙酰胆碱12或乙酰甲胆碱13处理之后。在这种方法中，只有肺的一小部分是一个微观alyzed，但一个研究报告指出，在PCLS气管收缩的测量从片仅改变约10％至切片，并且该差异是比得上在完整动物14使用肺功能测试看出。其他研究者使用PCLS作为离体方法用LPS 15温育后，研究在细胞因子的表达和细胞表面标志物的变化。 PCLS也已在小腺泡内动脉缺氧性肺血管收缩的离体模型中使用。这些容器位于不能使用其它的程序，包括从解剖动脉区段或胸膜下的血管16的分析记录到达肺的一部分。我们的实验室已主要用于PCLS可视化在稳定状态中活肺组织免疫细胞定位和以下的体内炎症刺激。这些都是我们的这个发展的过程如下。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Prox1-TdTomato transgenic mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td>018128</td>
			<td>B6;129S-Tg(Prox1-tdTomato)12Nrud/J</td>
		</tr>
		<tr>
			<td>OT-II OVA-specific TCR x Nur77-GFP transgenic mice</td>
			<td>Jackson Laboratory</td>
			<td>004194, 006617</td>
			<td>B6.Cg-Tg(TcraTcrb)425Cbn/J x C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J&#160;</td>
		</tr>
		<tr>
			<td>Rag1 knock-out mice</td>
			<td>Jackson Laboratory</td>
			<td>002216</td>
			<td>B6.129S7-Rag1tm1Mom/J</td>
		</tr>
		<tr>
			<td>Ovalbumin, Low Endo, Purified</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003059</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli</td>
			<td>Sigma-Aldrich Co.</td>
			<td>L2630-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing (Non-Sterile) 100 ft</td>
			<td>BD Diagnostic Systems</td>
			<td>427421</td>
			<td>0.86 mm inside diameter, 1.27 mm outside diameter</td>
		</tr>
		<tr>
			<td>GeneMate Sieve GQA Low Melt Agarose</td>
			<td>BioExpress</td>
			<td>E-3112-125</td>
			<td>2% solution dissolved in PBS at 70 &#176;C and held at 40 &#176;C.</td>
		</tr>
		<tr>
			<td>Compresstome VF-300</td>
			<td>Precisionary Instruments, Inc.</td>
			<td>VF-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Edge Stainless Razor Blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td>Disposable; 250/box. Blade should be changed for every lung.</td>
		</tr>
		<tr>
			<td>Krazy Glue All Purpose Instant Gel</td>
			<td>VWR</td>
			<td>500033-484</td>
			<td>Commonly available for $3/tube in local drugstores&#160;</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rat Serum</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>012-000-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse Serum</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>015-000-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Buffer</td>
			<td>Made in House</td>
			<td>N/A</td>
			<td>PBS w/ 0.5% bovine serum albumin, 0.1% NaN3, pH 7.4</td>
		</tr>
		<tr>
			<td>Fc Blocker (anti-CD16/32 antibodies)</td>
			<td>Made in House</td>
			<td>N/A</td>
			<td>Supernatant of cultured hybridoma cell line 2.4G2</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b eFluor 450</td>
			<td>eBioscience</td>
			<td>48-0112-80</td>
			<td>Anti-mouse CD11b eFluor 450 (clone: M1/70)&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c Brilliant Violet 605</td>
			<td>BioLegend</td>
			<td>101237</td>
			<td>Brilliant Violet 605 anti-mouse CD11c (clone: M1/70)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c Phycoerythrin</td>
			<td>eBioscience</td>
			<td>12-0114-82</td>
			<td>PE conjugated anti-mouse CD11c (clone: N418)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c Allophycocyanin</td>
			<td>BD Phamingen</td>
			<td>550261</td>
			<td>APC-labeled anti-mouse CD11c 9clone: HL3)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD88 Phycoerythrin</td>
			<td>BioLegend</td>
			<td>135806</td>
			<td>PE anti-mouse CD88 (clone: 20/70)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD103 Allophycocyanin</td>
			<td>eBioscience</td>
			<td>17-1031-82</td>
			<td>Anti-mouse CD103 APC (clone: 2E7)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD90.2/Thy1.2 eF450</td>
			<td>eBioscience</td>
			<td>48-0902-82</td>
			<td>Anti-mouse CD90.2 eFluor 450 (clone: 53-2.1)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD172a/Sirp1a Allophycocyanin</td>
			<td>eBioscience</td>
			<td>17-1721-82</td>
			<td>Anti-mouse CD172a APC (clone: P84)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD324 Brilliant Violet 421</td>
			<td>BD Horizon</td>
			<td>564188</td>
			<td>BV421 mouse anti-E Cadherin (clone: 5E8 also known as 5E8-G9-B4)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD324 Alexa Fluor 488</td>
			<td>eBioscience</td>
			<td>53-3249-82</td>
			<td>Anti-CD324(E-Cadherin) Alexa Flour 488 (clone: DECMA-1)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD324 Alexa Fluor 647</td>
			<td>eBioscience</td>
			<td>51-3249-82</td>
			<td>Anti-CD324(E-Cadherin) Alexa Flour 647 (clone: DECMA-1)</td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes 35mm petri dish, 14mm Microwell, No. 1.5 coverglass</td>
			<td>MatTek Corperation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered Coverglass</td>
			<td>ThermoFisher Scientific</td>
			<td>155411PK&#160;</td>
			<td>Pack of 16</td>
		</tr>
		<tr>
			<td>15 mm Coverslip, No. 1.5 Glass Thickness</td>
			<td>MatTek Corperation</td>
			<td>PCS-1.5-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Bare Platinum Wire</td>
			<td>World Precision Instruments</td>
			<td>PTP201</td>
			<td>0.020&#34; (0.5mm) diameter cut into ~1 cm long pieces and bent into an &#34;L&#34; shape</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36934</td>
			<td>Keep at 4 &#176;C, warm to room tempterature before use.</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, *LDEV-Free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss 880 multi-photon laser-scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Zen Black software version 8.1, 2012 (Zeiss)</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 20x/0.8 M27 objective lends</td>
			<td>Carl Zeiss</td>
			<td>420650-9901-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

precision-cut mouse lung slices to visualize live pulmonary dendritic cells

过敏原和病原体的吸入引起的各种肺免疫细胞类型的多个变化。流式细胞术是用于在免疫细胞上的细胞表面蛋白的定量分析的强大技术，但它提供对肺中这些细胞的定位和迁移模式的信息。类似地，趋化实验可以被执行以研究细胞的潜力， 在体外趋化因子反应，但是这些测定法不再现完整肺的复杂的环境。相对于这些前述技术中，肺中的个别细胞类型的位置可以容易地通过产生精确切割肺片（PCLS）中，用商购的，荧光标记的抗体染色它们，并通过共聚焦显微镜可视化部分可视化。 PCLS可以用于活的和固定的肺组织，和片可以涵盖区域一样大的整个叶的横截面。我们已经使用该协议成功地可视化的各种各样的肺细胞类型，包括不同类型的树突状细胞的巨噬细胞，嗜中性粒细胞，T细胞和B细胞，以及结构的细胞，例如淋巴，内皮的位置，和上皮细胞。可视化细胞相互作用，如树突状细胞和T细胞，在活的，三维的肺组织之间的能力，可以揭示细胞肺内如何移动，并与在稳定状态下和在炎症期间彼此相互作用。因此，与其他的方法，例如流式细胞术和定量PCR组合使用时，PCLS可以向背后肺的变应性和炎症性疾病的细胞事件的全面理解。

为了识别两个DC亚群从C57BL / 6小鼠中的位置，CD11b的喜 cDC上和CD103 + cDC上，PCLS切下并用特异于的CD11c单克隆抗体（mAb）染色，CD88，CD103，CD324和（E-钙粘蛋白）。抗体染色CD324气道上皮细胞，和CD88显示在巨噬细胞和嗜中性粒细胞，但不是cDC上8。这使我们能够从表面CD11c +巨噬细胞区分cDC上，并观察各细胞类型的气道（ 图1）</str...

Watch this Scientific Journal Video about Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells at JoVE.com

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

我们描述了用于产生精密切割肺片（PCLS）和免疫它们以显现各种免疫细胞类型的定位在肺中的方法。我们的协议可以扩展在各种条件下可视化的位置和许多不同细胞类型的功能。

精密切割的小鼠肺切片可视化现场肺树突状细胞

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

precision-cut-mouse-lung-slices-to-visualize-live-pulmonary-dendritic

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  National Institute of Environmental Health Sciences, NIH. 我们描述了用于产生精密切割肺片（PCLS）和免疫它们以显现各种免疫细胞类型的定位在肺中的方法。我们的协议可以扩展在各种条件下可视化的位置和许多不同细胞类型的功能。 

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

Isolation of Mouse Lung Dendritic Cells

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

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

小鼠肺树突状细胞的分离

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

科研

免疫与感染

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

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

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

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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 ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

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 ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

嗜肺军团菌外膜囊泡:隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

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

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

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

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

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

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

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

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

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

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

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