Name: flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies
Uploaded: 2012-12-26T12:00:00
Description: 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

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?...

<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>

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 ...

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

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

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

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

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

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

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

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

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

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

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

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 ...