我们要感谢M.Höxter的排序的主要鼠AECII从生物安全2级样品的技术援助。 这项工作是支持的补助金从德国研究基金会（DFG）DB（SFB587，TP B12和BR2221/1-1的的）和的津贴从汉诺威生物医学研究学院（DFG GSC 108）AA。 DB支持总统的倡议下合同编号W2/W3-029德国研究中心，亥姆霍兹联合会（HGF）和网络基金。

关于所需的试剂和材料的细节在表中列出的，在端部的下面的协议。在开始工作之前，准备15毫升管（每一个鼠标），其中包含4毫升分离酶和预暖至37℃的水浴中。不久，在一个加热块1％低熔点琼脂糖（在水中）的小等分试样加热至95℃，直至液化并随后冷却至45℃，直至使用。 1。制备小鼠肺<ol><li>牺牲鼠标的CO 2窒息。注意:不要进行颈椎错位，因为这将损害气管，使协议的步骤，如，如安装的液体肺，不能成功进行。确保伤害反射的损失。 </li><li>喷用乙醇和鼠标，使一个长期的切割，沿腹正中线的身体。拉腹部的皮毛和皮肤，随后还精心削减和删除腹膜的。 </li><li>抽血的切割的左和右颈静脉（ 腔jugularis）以及肾动脉（ 动脉renalis）的小鼠。拆下流动的血液与组织。 </li><li>小心穿刺，然后取出膜片，暴露心脏和肺切掉肋骨。要特别小心，不要伤到肺。 </li><li>有了一个26G插管和10毫升注射器充满冷的磷酸盐缓冲盐水（PBS），穿刺的心脏的右心室，直到无血用PBS灌注肺。 </li><li>剪切和删除的唾液腺暴露气管。此外，小心切气管周围的肌肉。 </li><li>将一个22G留置气管插管插入，拔出针头，并推动对肺部的塑料导管。修复的导管，通过把一小块纱线围绕气管和导管。 </li></ol> 2。肺组织的酶消化如果需要的话，支气管肺泡灌洗液样品可以是制备通过在气管导管插入下一步骤之前，用PBS或培养基冲洗肺。 <ol><li>使用一个2毫升的注射器，小心地注入2毫升分散酶（从4毫升）的最大体积的通过导管进入肺，使得所有裂片完全展开。交换含有0.5毫升液化琼脂糖用1ml注射器和注射器也进入肺灌输。 </li><li>将注射器留在导管上，以防止倒流琼脂糖并立即与实验室薄纸和冰覆盖肺。让琼脂糖凝胶一两分钟。 </li><li>取出纸巾，冰，注射器，导管，切断气管和消费的肺，心脏和胸部的胸腺。然后在培养皿中，用PBS冲洗肺，取出心脏，胸腺，其余气管。 </li><li>将肺成剩余的2毫升分散酶，并在室温下孵育45分钟。 </li></ol> 3。制备的龙细胞悬浮<ol><li>取出肺从分散酶，并将其转移到一碟含7毫升的anti-CD16/32抗体的（1μg/ ml的最终浓度）在DMEM培养基和100μlDNA酶。 </li><li>使用镊子，完全瓦解拉它除了肺组织，并在室温下孵育10分钟，同时轻轻摇晃摇杆设置为200转。如果需要的话，然后可以被存储在细胞悬浮液在4℃下，直到下一个步骤。 </li><li>先用100微米，随后用70微米，48微米和30微米孔隙通过尼龙网过滤细胞悬浮液。照顾以及管和菜肴用DMEM彻底冲洗的各网格的单元格，以减少损失，并最大限度地提高产量。如果你正在汇集样品，这可以在这里实现，随后通过细胞悬浮液通过一组小鼠相同的过滤器。续约在堵塞的情况下的过滤器。 </li><li>根据卷上，将滤液转移到一个或多个50 ml离心管，并在160×g离心15分钟，4℃，离心除去上清液。 </li><li>在2ml红细胞裂解缓冲液（0.15 M NH 4 Cl的 ，0.01M KHCO 3，0.1mM的EDTA，pH值7.2）中重悬细胞沉淀，并迅速终止通过加入13毫升的DMEM培养基中的溶解。在160×g离心12分钟，4℃，将该溶液转移到一个15毫升的试管和离心</li></ol> 4。抗体染色流式细胞仪检测细胞分选<ol><li> 3毫升的主要抗体鸡尾酒，准备在DMEM培养基稀释适合流式细胞仪分析，重悬细胞。 （抗体先前为最佳工作稀释滴定进行染色1×10 6个脾细胞中的体积为100μl。使用为每个所用的抗体对细胞的最佳浓度的的初级抗体混合物含有的3毫升汇集来自五个小鼠的。如果有更多的老鼠被汇集到一个样本，调整音量）。 </li><li>染色，室温孵育10分钟，在黑暗中在4℃下</li><li>洗涤细胞填充15毫升DMEM培养基并离心管，在160×g离心12分钟，和4℃。 </li><li>如果耦合或生物素标记的抗体用于4.1:去上清，悬浮细胞在2 - 3毫升二次抗体鸡尾酒。在黑暗中染色10分钟，在4°C。 </li><li>通过填充管在160×g离心12分钟，4℃，用DMEM和离心来洗涤细胞</li><li>细胞重悬于1毫升DMEM和预过滤器中通过一个50微米的过滤器用于细胞分拣成管。可选:细胞计数，以获得总的在肺细胞悬浮液中的细胞数。 </li></ol> 5。细胞分选<ol><li>振荡排序前的细胞混合。使用100μm的喷嘴用于细胞分拣AECII。护套的流体压力，以及激光发射和检测波长依赖于所用的仪器为流式细胞仪细胞分选的荧光染料，以及用于在antib全球最著名的染色过程。 </li><li>门上的SSC的高细胞中，所使用的荧光染料染色和排序管DMEM是否定的。使用前向散射的高度（FSC-H） - 区域（FSC-A）和侧向散射的高度（SSC-H）与面积（SSC-A）窗口，以排除任何双峰或细胞聚集体（详见门控策略图1）。 </li><li>为了收集细胞，离心20分钟，在280 XG和4°C。 </li><li>重悬细胞培养介质或缓冲所需的后续分析。 </li></ol>

<ol>
	<li>Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelial+type+II+cell:+defender+of+the+alveolus+revisited.">Alveolar epithelial type II cell: defender of the alveolus revisited.</a> Respir. Res. 2, 33-46 (2001).</li><li>Folkerts, G., Nijkamp, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airway+epithelium:+more+than+just+a+barrier.">Airway epithelium: more than just a barrier.</a> Trends Pharmacol. Sci. 19, 334-341 (1998).</li><li>Gereke, 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=Phenotypic+alterations+in+type+II+alveolar+epithelial+cells+in+CD4++T+cell+mediated+lung+inflammation.">Phenotypic alterations in type II alveolar epithelial cells in CD4+ T cell mediated lung inflammation.</a> Respir. Res. 8, 47 (2007).</li><li>Gereke, M., Jung, S., Buer, J., Bruder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+type+II+epithelial+cells+present+antigen+to+CD4(+)+T+cells+and+induce+Foxp3(+)+regulatory+T+cells.">Alveolar type II epithelial cells present antigen to CD4(+) T cells and induce Foxp3(+) regulatory T cells.</a> Am. J. Respir. Crit Care Med. 179, 344-355 (2009).</li><li>Gribar, S. C., Richardson, W. M., Sodhi, C. P., Hackam, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+longer+an+innocent+bystander:+epithelial+toll-like+receptor+signaling+in+the+development+of+mucosal+inflammation.">No longer an innocent bystander: epithelial toll-like receptor signaling in the development of mucosal inflammation.</a> Mol. Med. 14, 645-659 (2008).</li><li>Herold, 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=Alveolar+epithelial+cells+direct+monocyte+transepithelial+migration+upon+influenza+virus+infection:+impact+of+chemokines+and+adhesion+molecules.">Alveolar epithelial cells direct monocyte transepithelial migration upon influenza virus infection: impact of chemokines and adhesion molecules.</a> J. Immunol. 177, 1817-1824 (2006).</li><li>Knight, D. A., Holgate, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+airway+epithelium:+structural+and+functional+properties+in+health+and+disease.">The airway epithelium: structural and functional properties in health and disease.</a> Respirology. 8, 432-446 (2003).</li><li>Schmiedl, A., Kerber-Momot, T., Munder, A., Pabst, R., Tschernig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+distribution+in+lung+parenchyma+early+after+pulmonary+infection+with+Pseudomonas+aeruginosa.">Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa.</a> Cell Tissue Res. 342, 67-73 (2010).</li><li>Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., Jean, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal-+and+Strain-Specific+Host+MicroRNA+Molecular+Signatures+Associated+with+Swine-Origin+H1N1+and+Avian-Origin+H7N7+Influenza+A+Virus+Infection.">Temporal- and Strain-Specific Host MicroRNA Molecular Signatures Associated with Swine-Origin H1N1 and Avian-Origin H7N7 Influenza A Virus Infection.</a> J. Virol. 86, 6109-6122 (2012).</li><li>Marriott, H. 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=Interleukin-1beta+regulates+CXCL8+release+and+influences+disease+outcome+in+response+to+Streptococcus+pneumoniae,+defining+intercellular+cooperation+between+pulmonary+epithelial+cells+and+macrophages.">Interleukin-1beta regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages.</a> Infect. Immun. 80, 1140-1149 (2012).</li><li>Mata, M., Morcillo, E., Gimeno, C., Cortijo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetyl-L-cysteine+(NAC)+inhibit+mucin+synthesis+and+pro-inflammatory+mediators+in+alveolar+type+II+epithelial+cells+infected+with+influenza+virus+A+and+B+and+with+respiratory+syncytial+virus+(RSV).">N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV).</a> Biochem. Pharmacol. 82, 548-555 (2011).</li><li>Zarbock, 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=The+surfactant+protein+C+mutation+A116D+alters+cellular+processing,+stress+tolerance,+surfactant+lipid+composition,+and+immune+cell+activation.">The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.</a> BMC. Pulm. Med. 12, 15 (2012).</li><li>Taubenberger, J. K., Morens, 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=The+pathology+of+influenza+virus+infections.">The pathology of influenza virus infections.</a> Annu. Rev. Pathol. 3, 499-522 (2008).</li><li>Dobbs, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+alveolar+type+II+cells.">Isolation and culture of alveolar type II cells.</a> Am. J. Physiol. 258, L134-L147 (1990).</li><li>Corti, M., Brody, A. R., Harrison, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+murine+alveolar+type+II+cells.">Isolation and primary culture of murine alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 14, 309-315 (1996).</li><li>Beers, M. F., Kim, C. Y., Dodia, C., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+synthesis,+and+processing+of+surfactant+protein+SP-C+in+rat+lung+analyzed+by+epitope-specific+antipeptide+antibodies.">Localization, synthesis, and processing of surfactant protein SP-C in rat lung analyzed by epitope-specific antipeptide antibodies.</a> J. Biol. Chem. 269, 20318-20328 (1994).</li><li>Phelps, D. S., Floros, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+pulmonary+surfactant+proteins+using+immunohistochemistry+and+tissue+in+situ+hybridization.">Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization.</a> Exp. Lung Res. 17, 985-995 (1991).</li><li>Wang, 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=Differentiated+human+alveolar+type+II+cells+secrete+antiviral+IL-29+(IFN-lambda+1)+in+response+to+influenza+A+infection.">Differentiated human alveolar type II cells secrete antiviral IL-29 (IFN-lambda 1) in response to influenza A infection.</a> J. Immunol. 182, 1296-1304 (2009).</li><li>Wang, 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=Innate+immune+response+to+influenza+A+virus+in+differentiated+human+alveolar+type+II+cells.">Innate immune response to influenza A virus in differentiated human alveolar type II cells.</a> Am. J. Respir. Cell Mol. Biol. 45, 582-591 (2011).</li></ol>

没有利益冲突的声明。

用流式细胞术隔离的小鼠AECII我们的协议提供了一个快速的方式访问主要的小鼠肺细胞的功能和分子生物学研究的整个范围。所描述的程序产生非常可行的，纯粹的人口AECII足够的数量，为的直接后续分析，如RNA分离（ 见图2b）和转录组的研究。对于功能性的应用程序，它也可以培养分离的细胞，允许， 例如生成AECII条件培养基或共培养实验。作为一个好处，尤其是对这些功能?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>留置套管Introcan 22G </td><td>布劳恩</td><td> REF 4252098B </td><td></td></tr><tr><td>中性蛋白酶，100毫升（5000酪蛋白单位） </td><td> BD Biosciences公司</td><td> 354235 </td><td> 〜4毫升15毫升管分装，储存在-20°C </td></tr><tr><td> Biozym斑块琼脂糖</td><td> Biozym </td><td> 840101 </td><td> 1％w / v在H 2 O </td></tr><tr><td>从牛胰腺，2000年Kunitz型单位/瓶的脱氧核糖核酸酶Ⅰ </td><td> Sigma-Aldrich公司</td><td> D4263 </td><td>内容1瓶新鲜溶解在300μLDMEM </td></tr><tr><td> DMEM </td><td> Gibco公司</td><td> 22320-022 </td><td>作为制造商（低Glucose，丙酮酸，HEPES） </td></tr><tr><td>细胞粗滤器（100微米，75微米） </td><td> BD猎鹰</td><td> 352360，352350 </td><td></td></tr><tr><td>尼龙筛（48微米，30微米） </td><td> Bückmann有限公司</td><td> 03-48/26-1020，03-30/18-108 </td><td></td></tr><tr><td> CellTrics 50微米的过滤器</td><td> PARTEC </td><td> 04-0042-2317 </td><td></td></tr><tr><td>抗小鼠CD16/CD32 </td><td> BioLegend </td><td> 101302 </td><td>克隆93;纯化</td></tr><tr><td>的抗小鼠F4/80 </td><td> BioLegend </td><td> 123116 </td><td>克隆的BM8; APC耦合</td></tr><tr><td>抗小鼠CD11b的</td><td> BioLegend </td><td> 101208 </td><td>克隆M1/70，PE耦合</td></tr><tr><td>抗鼠的CD11c </td><td> BioLegend </td><td> 117310 </td><td>克隆N418，APC耦合</td></tr><tr><td>抗小鼠CD45 </td><td> BioLegend </td><td> 103102 </td><td>克隆30-F11;纯化</td></tr><tr><td>抗鼠CD19 </td><td> eBioscience公司</td><td> 12-0193-83 </td><td>生物质1D3，PE耦合</td></tr><tr><td>的多克隆山羊抗大鼠IgG </td><td> BD Pharmingen公司</td><td> 550767 </td><td>多克隆抗体，PE耦合</td></tr><tr><td></td><td></td><td></td><td>耦合的替代荧光染料的抗体可用于，根据流式细胞仪和激光提供。 </td></tr></tbody></table>

flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies

纵观过去几年，各方面的免疫调节肺的肺泡Ⅱ型上皮的细胞（AECII）的贡献已被越来越多的认可。 AECII已显示出参与在发炎的气道的细胞因子的产生，甚至作为抗原呈递细胞在感染和T-细胞介导的自身免疫1-8。因此，他们也特别有趣的气道高反应性的临床环境下，比如外资和自身抗原以及感染，直接或间接地针对AECII。然而，我们了解的详细服务的II型肺泡上皮细胞在肺的健康以及在炎症的免疫功能仍然是零碎的。许多的研究AECII功能是使用鼠标或9-12人肺泡上皮细胞株。工作与细胞系确实提供了一系列好处，如提供大量ØF细胞广泛的分析。然而，我们相信使用的主要鼠AECII允许更好地了解复杂的过程，如感染或自身免疫性炎症的作用，这种细胞类型中。初级的的小鼠AECII可以直接从动物等呼吸系统疾病的痛苦，这意味着他们已经给所有其他的外在因素发挥作用的分析设置隔离。作为一个例子，可行的AECII可以分离出小鼠滴鼻感染A型流感病毒，主要目标，这些细胞的复制13。更重要的是，通过从健康小鼠中分离的AECII 体外感染，安装在感染的细胞的反应的研究可以进一步延长。 是根据我们的协议进行的原发性小鼠AECII的隔离的小鼠肺然后通过标记所得到的细胞悬浮液与特异性抗体的CD11c，CD11b和F4/80，酶消化，CD19，CD45和CD16/CD32。颗粒AECII然后确定为未标记的和侧向散射（SSC 高 ）细胞种群，并通过荧光激活细胞分选3被分离。 隔离小鼠肺上皮细胞的替代方法相比，我们的协议流式细胞仪分离的AECII负选择在比较短的时间内产生不动，高度可行的和纯AECII的。此外，和在常规的隔离方法，通过平移和淋巴细胞耗竭相反通过结合抗体耦合磁珠14，15，流式细胞仪细胞分选允许通过细胞大小和粒度歧视。由于仪器流式细胞仪细胞分选是可用的，所描述的程序，可以应用于在相对低的成本。下一步标准的抗体和酶肺解体，不需要额外的试剂，如磁珠是必需的。分离的细胞是适用于范围广泛的功能和分子生物学研究，其中包括在体外培养细胞和T细胞刺激实验，以及转录组，蛋白质组或分泌的分析3，4。

排序时从健康小鼠肺细胞悬浮液的分离，AECII门通常会占到约42±10％的所有事件。这个百分比可以是明显降低用于呼吸系统疾病，如病毒感染的小鼠时，作为初始的细胞悬浮液，将含有相当高的比例，淋巴细胞和其他免疫细胞的招募呼吸道。有关AECII分离从在第3天以下感染IAV感染肺部中，我们已经观察到细胞在AECII门的频率减少了大约50％。 一个典型的排序和重新排序的细胞?...

Watch this Scientific Journal Video about Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies at JoVE.com

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

我们描述了迅速隔离的主要鼠Ⅱ型肺泡上皮的细胞（AECII）流式细胞仪的负选择。这些AECII显示活力和纯度高，适用于范围广泛的功能和分子生物学研究，他们的作用，自身免疫性疾病或感染性疾病，如呼吸系统疾病。

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

flow-cytometric-isolation-primary-murine-type-ii-alveolar-epithelial

Research

JoVE Journal

Immunology and Infection

26.2K 次观看.  Helmholtz Centre for Infection Research. 我们描述了迅速隔离的主要鼠Ⅱ型肺泡上皮的细胞（AECII）流式细胞仪的负选择。这些AECII显示活力和纯度高，适用于范围广泛的功能和分子生物学研究，他们的作用，自身免疫性疾病或感染性疾病，如呼吸系统疾病。

视频: Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

科研

免疫与感染

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

中性粒细胞衍生微粒的分离与鉴定功能研究

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...