1。之前的过程中，准备和收集下列项目<ol><li>请参考图1a中的形象）和1b为建设一个木制的平台，以抑制在操作过程中鼠标）。 </li><li>消化混合淋巴结 - 的HBSS + 1.25mg/ml胶原酶TypeIV或2.5 mg / mL胶原酶ð </li><li>消化肺部混合 - 的HBSS + 1 mg / mL胶原酶</li><li>稀释在PBS腹腔注射乳胶珠（1:20）的LDLNs可视化</li></ol> 注：LDLNs很小，很难找到。要了解如何找到他们，注入200μL稀释的红色荧光乳胶珠到鼠标的IP 1:20。腹膜水渠这些LDLNs 8。 24 HPI，暴露胸腔上面所述的鼠标。 LDLNs会出现粉红色的珠子从腹腔排水。 <ol start="5"><li>红细胞（RBC）的细胞裂解液- 0.15米的NH 4 CL，10MM KHCO 3，0.1毫米NA 2 EDTA 1L DH 2 0 。调整pH值到7.2-7.4，并在室温下保存。 </li><li> 70μm或100μm的细胞过滤器</li><li>商业解决方案0.5M EDTA，pH值= 8.0 </li><li> 氯化钙和氯化镁2的HBSS无</li><li>剪刀钝端钳</li><li>喉镜</li><li>灌胃针（22号）弯曲的角度（图2） </li><li>聚苯乙烯泡沫塑料板和传播鼠标引脚</li><li>顶盘天平</li><li>苯胺蓝溶液- 10mg/ml的H 2 O </li></ol> 注：我们将使用这一解决方案，为它注入技术的示范。 2。鼠标麻醉的制备和管理<ol><li>在无菌PBS准备甲苯噻嗪和氯胺酮的混合物，使终浓度2mg/ml和10mg/ml的分别。 </li><li>称量鼠标顶部泛平衡。 </li><li>计算每个鼠标所需的剂量。在甲苯噻嗪（2-4毫克/公斤），氯胺酮（70-80毫克/公斤）体重的剂量使用的准备混合物适当的体积和注入intaperitoneally。每个小鼠品系可能需要麻醉剂的优化。接受过多麻醉的小鼠可能会​​降低呼吸速率，接种灌输到肺部后，可能是致命的。 </li><li> 3-5分钟后，观察麻醉效果的鼠标。下列标志的检查，以确认鼠标是完全麻醉： <ol><li>呼吸速度应放慢。 </li><li>鼠标不应延伸其手臂，颈部时捞起。 </li><li>不应该有任何后肢刺激时的反应。 如果这些条件不能满足，等待几分钟，然后再次检查，然后再继续下一步。 </li></ol></li></ol> 3。气管内注射<ol><li>准备在50μL生理盐水innoculum。填充1ml注射器装用无菌弯曲与innoculum灌胃针。背后的innoculum100μL空气袋应装入注射器，以确保所有的液体注入到肺。 </li><li>广场上倾斜的木平台，其门牙挂在电线上的鼠标轻轻地抑制在一块色带。 </li><li>打开喉镜，用左手（如果你是右撇子）和抢一双钝结束钳。使用喉镜和镊子的尖端轻轻撬开口。 </li><li>随着钳，拉舌，抱到一边。指南对口腔后部的喉镜片。保持在90度角，轻轻地按下，直到你看到气管开放喉镜。握住喉镜到位。时从嘴里拉出舌头和应做轻轻的发生，使无组织损伤，应该小心。 </li><li>用另一只手，采取包含innoculum，你的右手弯曲灌胃针装有1ml注射器，插入气管针，弯针，直到前门牙。均匀推柱塞提供innoculum。 innoculum应该没有泡沫。尽快拉出气管针。老鼠窒息死亡，如果时间过长阻塞气管。按住鼠标几秒钟直立允许进入肺部吸入innoculum。 </li><li>鼠标的平台和地点附近的一个取暖灯。不要直接加热下离开小鼠长时间。鼠标应在30分钟内完全清醒后的程序（恢复时间可能会有所不同的基础上株）。在此期间，应努力观察小鼠和不应该允许太热或太冷，这可能也把到呼吸窘迫导致死亡的小鼠。 </li></ol> 4。采伐和加工的龙引流淋巴结和肺流式细胞仪<ol><li>牺牲小鼠使用所需的方法，为每AVMA安乐死指引。这可能会有所不同，取决于个人收获的成果。致命的钠不巴比妥管理是一个合适的安乐死程序的一个例子。我们还没有看到的CO 2安乐死的负肺效应。 </li><li>引脚的上夹层板的胳膊和腿。使切口，沿中间用剪刀腹侧。轻轻拉动皮肤暴露胸壁的肌肉。 </li><li>通过胸壁肌肉的切口，暴露腹部器官。 </li><li>按住胸骨尖镊子和穿刺隔膜。切沿左肋双方的隔膜。 </li><li>用剪刀夹层板表面平行，穿过沿​​背腹线两侧胸腔。 </li><li>稍微解除左肋。在右侧的心，只是下面胸腺，寻找两个淋巴结。一个稍大的第三个淋巴结是位于下方的小动脉血管，气管垂直运行。收获淋巴结肿大，淋巴结消化混合成1毫升。 </li><li>收获后淋巴结肿大，与一双镊子的心脏和抓住与另一肺部。收获到冰冷的PBS3毫升肺在6孔板。 </li><li>独立的肺部叶，吸出PBS及使用镊子和剪刀切成小块耳垂。添加肺部消化混合3毫升。在这一步，保持6孔板上的冰。 </li><li> 25-30分钟和30分钟的肺部淋巴结在37 ° C。加入EDTA至终浓度为10mm消化混合物停止反应，并置于冰上。 </li><li>使用3毫升注射器，通过18GA针肺片的3倍。捣碎件通过70 -100μm的细胞过滤器使用一个1ml注射器柱塞回结束的肺部和淋巴结。洗净无钙的HBSS10毫升细胞过滤器+和Mg 2 +。在500 XG离心5分钟。 </li><li> 5毫升红细胞裂解液重悬沉淀和孵化在室温下3分钟。添加无钙的HBSS10毫升+和Mg 2 +淬火溶解。在500 XG离心5分钟。调出肺癌和LDLN单细胞悬液，分别至10ml和5毫升。使用一个hemacytometer活细胞计数台盼蓝拒。细胞现在已经准备好，染色流式细胞仪。 1叶肺，可能是足够的流式细胞仪。其他叶可用于其他实验问题，在相同的鼠标，如细胞因子或组织学组织匀浆。 </li></ol> 5。代表性的成果： 最不发达国家和在肺部的肺泡巨噬细胞获得抗原从肺部的肺泡，紧随其后注射。图3显示的CD11c +（表示对最不发达国家和肺泡巨噬细胞）的百分比细胞，荧光标记的炭疽杆菌孢子积极，30分钟后，注射。 〜15.9％，CD45 +的CD11c +细胞肺癌单细胞悬液，在这个时候，包庇炭疽杆菌孢子（图3，下板）。在肺部的CD11c +细胞的进一步鉴定，是指金正日和Braciale 4 。经常使用荧光标记的珠子，以评估贩卖颗粒抗原LDLNs 9 。为了评估这从肺部LDLNs运输，荧光乳胶珠注射LPS和24 HPI分析LDLNs。约4％的CD11c 您好细胞的LDLNs包庇24hpi（图4）的荧光珠。这些代表性的实验表明，这个协议可以有效地用于跟踪收购吸入颗粒抗原的细胞在肺部，随后交通的LDLNs。 <img src="/files/ftp_upload/2702/2702fig1.jpg" alt="图1"/> 图1。在它注射抑制小鼠的木制平台 。木块在一个角度切入，金属栏杆安装，前视图图1a）和侧视图图1b） 。伤口周围的金属条电线的一小片，锚鼠标的前门牙。一块色带连接到平台，围绕鼠标配合到位，在手术过程中保持。 <img src="/files/ftp_upload/2702/2702fig2.jpg" alt="图2"/> 图2。它注射针灌胃。灌胃针（右）〜135 °（左）为它注入的角度弯曲。 <img src="/files/ftp_upload/2702/2702fig3.jpg" alt="图3"/> 图3。所有海港注入绿色荧光蛋白表达炭疽杆菌 （星级p6GFP），5小时后的挑战肺部表面CD11c +细胞的百分比。 AJ。小鼠用1X的挑战在5小时后感染10 8 GFP的炭疽杆菌孢子和肺部进行了分析。点图显示的CD11c百分比+肺炭疽杆菌阳性的细胞， 。 <img src="/files/ftp_upload/2702/2702fig4.jpg" alt="图4"/> 图4。 （黄/绿）在肺癌淋巴结我... ...吨注射24小时后检测荧光珠 。 AJ小鼠给予10μg脂多糖（右图）和LDLNs分析24hpi要么PBS（左侧面板）或5X10 9 Fluoresbrite永贵微球（0.5微米）。代表点图显示在LDLNs海港荧光珠对绘制一个空通道的CD11c 您好细胞的百分比。

<ol>
	<li>Grayson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+lung+dendritic+cell+maturation+and+migration+during+respiratory+viral+infection.">Controls for lung dendritic cell maturation and migration during respiratory viral infection.</a> J Immunol. 179, 1438-1438 (2007).</li><li>GeurtsvanKessel, C. H., Lambrecht, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Division+of+labor+between+dendritic+cell+subsets+of+the+lung.">Division of labor between dendritic cell subsets of the lung.</a> Mucosal Immunol. 1, 442-442 (2008).</li><li>Bar-Haim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrelationship+between+dendritic+cell+trafficking+and+Francisella+tularensis+dissemination+following+airway+infection.">Interrelationship between dendritic cell trafficking and Francisella tularensis dissemination following airway infection.</a> PLoS Pathog. 4, e1000211-e1000211 (2008).</li><li>Kim, T. S., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+dendritic+cell+subsets+differ+in+their+capacity+to+support+the+induction+of+virus-specific+cytotoxic+CD8++T+cell+responses.">Respiratory dendritic cell subsets differ in their capacity to support the induction of virus-specific cytotoxic CD8+ T cell responses.</a> PLoS One. 4, e4204-e4204 (2009).</li><li>Bakocevic, N., Worbs, T., Davalos-Misslitz, A., Forster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-dendritic+cell+interaction+dynamics+during+the+induction+of+respiratory+tolerance+and+immunity.">T cell-dendritic cell interaction dynamics during the induction of respiratory tolerance and immunity.</a> J Immunol. 184, 1317-1317 (2010).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+particle+size+on+the+pathology+and+efficacy+of+vaccination+in+a+murine+model+of+inhalational+anthrax.">Influence of particle size on the pathology and efficacy of vaccination in a murine model of inhalational anthrax.</a> J Med Microbiol. , (2010).</li><li>Kiyono, H., Fukuyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NALT-+versus+Peyer's-patch-mediated+mucosal+immunity.">NALT- versus Peyer's-patch-mediated mucosal immunity.</a> Nat Rev Immunol. 4, 699-699 (2004).</li><li>Parungo, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+drainage+of+the+peritoneal+space:+a+pattern+dependent+on+bowel+lymphatics.">Lymphatic drainage of the peritoneal space: a pattern dependent on bowel lymphatics.</a> Ann Surg Oncol. 14, 286-286 (2007).</li><li>Jakubzick, C., Helft, J., Kaplan, T. J., Randolph, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+methods+to+study+pulmonary+dendritic+cell+migration+reveals+distinct+capacities+of+DC+subsets+to+acquire+soluble+versus+particulate+antigen.">Optimization of methods to study pulmonary dendritic cell migration reveals distinct capacities of DC subsets to acquire soluble versus particulate antigen.</a> J Immunol Methods. 337, 121-121 (2008).</li><li>Higgins, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+levels+of+M-CSF+and+GM-CSF+influence+the+specific+generation+of+macrophage+populations+during+infection+with+Mycobacterium+tuberculosis.">Relative levels of M-CSF and GM-CSF influence the specific generation of macrophage populations during infection with Mycobacterium tuberculosis.</a> J Immunol. 180, 4892-4892 (2008).</li><li>Kool, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alum+adjuvant+boosts+adaptive+immunity+by+inducing+uric+acid+and+activating+inflammatory+dendritic+cells.">Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.</a> J Exp Med. 205, 869-869 (2008).</li><li>Costa, D. L., Lehmann, J. R., Harold, W. M., Drew, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transoral+tracheal+intubation+of+rodents+using+a+fiberoptic+laryngoscope.">Transoral tracheal intubation of rodents using a fiberoptic laryngoscope.</a> Lab Anim Sci. 36, 256-256 (1986).</li><li>Takahashi, S., Patrick, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+lymphatic+drainage+to+individual+thoracic+and+cervical+lymph+nodes+in+the+rat.">Patterns of lymphatic drainage to individual thoracic and cervical lymph nodes in the rat.</a> Lab Anim. 21, 31-31 (1987).</li><li>Broeck, W. V. a. n. d. e. n., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+Descriptive+study+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.</a> J Immunol Methods. 312, 12-12 (2006).</li></ol>

我们利用这个协议，从肺到LDLNs 研究炭疽杆菌孢子的贩运。对于类似的应用程序，传送到肺部的颗粒数应慎重选择，使注射材料可通过流式细胞仪检测的LDLNs。我们还成功地使用这种方法为具体的肺细胞用荧光标记抗体的人口的细胞和标签的收养转移。此外，这种方法是经常使用的管理直接进入肺部10博莱霉素（化学诱导的肺间质纤维化）和细胞因子。 尽管此?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 70μm细胞stariner </td><td>屋宇署</td><td></td><td></td></tr><tr><td>苯胺蓝</td><td>费舍尔</td><td> A967 - 25 </td><td></td></tr><tr><td>动物饲养针</td><td>波普尔和父子公司</td><td> 7920 </td><td></td></tr><tr><td>胶原酶</td><td>西格玛</td><td> C2139 </td><td></td></tr><tr><td>胶原酶TypeIV </td><td>沃辛顿</td><td></td><td></td></tr><tr><td>胶原酶ð </td><td>罗氏公司</td><td> 11088974103 </td><td></td></tr><tr><td> DPBS </td><td> Invitrogen公司</td><td> 14190 </td><td></td></tr><tr><td> Fluoresbrite永贵微（0.5微米） </td><td> Polysciences，公司</td><td> 17152 </td><td></td></tr><tr><td>氯化钙和氯化镁的HBSS无</td><td> Invitrogen公司</td><td> 14170 </td><td></td></tr><tr><td>盐酸氯胺酮（100mg/ml） </td><td>赫士睿公司</td><td></td><td></td></tr><tr><td>喉镜片</td><td> PennCentury，INC </td><td>型号LS - 1 </td><td>参考到<a href="http://www.penncentury.com">www.penncentury.com</a> </td></tr><tr><td>轻型纤维光纤喉镜</td><td> WelchAllyn </td><td> 80814 </td><td></td></tr><tr><td>红荧光珠（0.5微米） </td><td> Invitrogen公司</td><td> F8812 </td><td>对于腹腔注射</td></tr><tr><td>甲苯噻嗪（100mg/ml） </td><td>劳埃德实验室</td><td></td><td></td></tr></tbody></table>

non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry

连续采样在肺部的肺泡吞噬细胞，如肺泡巨噬细胞和肺树突状细胞的最不发达国家（LDCs）的抗原。最不发达国家，特别是被称为迁移到肺淋巴结（LDLNs）他们目前吸入各种免疫原1,2发起一个适当的免疫反应的T细胞抗原。肺和空气中的抗原在小鼠模型之间的相互作用，抗原滴鼻给药1,3,4，气管或气溶胶6。每条路线交货涉及不同的技术技能和限制，需要考虑设计一个实验之前。例如，鼻腔和雾化曝光提供抗原肺部和上呼吸道。因此，抗原可进入鼻相关淋巴组织（NALT）7，潜在的复杂结果的解释。此外，吞咽，打喷嚏和鼠标的呼吸频率，也可能导致在交付的剂量不一致。虽然上呼吸道的参与可能是一些研究的首选，它可以实验侧重于专门在肺部发起的事件复杂化。在这种背景下的气管（IT）的路线是可取的，因为它提供的测试材料，直接进入肺部，并绕过NALT。许多注射协议涉及要么盲目通过口腔或气管的手术暴露访问肺部气管插管。在这里，我们描述了一个简单的，一致的，它滴入的非手术方法。气管的开放是可视化使用喉镜和弯曲灌胃针插入进气管直接提供innoculum的。我们还描述了采伐和加工用流式细胞仪分析抗原贩运LDLNs和肺部的程序。

Watch this Scientific Journal Video about Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry at JoVE.com

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

我们说明进入肺部通过气管内麻醉小鼠的非手术治疗提供试验材料。这种方法允许肺暴露于细菌和病毒的病原体，细胞因子，抗体，珠子，化学品，染料。我们进一步描述采伐和肺和肺淋巴结（LDLNs），流式细胞仪检测处理。

非手术治疗的小鼠气管内滴入肺和肺淋巴结流式细胞仪分析

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

non-surgical-intratracheal-instillation-mice-with-analysis-lungs-lung

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Immunology and Infection

57.0K 次观看.  University of Colorado School of Medicine. 我们说明进入肺部通过气管内麻醉小鼠的非手术治疗提供试验材料。这种方法允许肺暴露于细菌和病毒的病原体，细胞因子，抗体，珠子，化学品，染料。我们进一步描述采伐和肺和肺淋巴结（LDLNs），流式细胞仪检测处理。

视频: Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

科研

免疫与感染

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

细胞内染色和流式细胞仪识别鼠主动脉，肾脏和淋巴结内淋巴细胞亚群的高血压模型

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

小鼠与人肺泡巨噬细胞的分离和体外培养

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

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

流式细胞术测定小鼠胸腺、胰腺干淋巴结和脾脏的调节 t 细胞亚群

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

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

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

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

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

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

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

单方支气管内酸灌输自我限制急性肺损伤模型

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...