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
Summary
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。
Abstract
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。
Protocol
Representative Results
Discussion
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?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of reagent | Company | Catalogue number | Comments |
| indwelling cannula Introcan 22G | Braun | REF 4252098B | |
| Dispase, 100 ml(5000 caseinolytic units) | BD Biosciences | 354235 | aliquot to 4 ml in 15 ml tubes, store at -20 °C |
| Biozym Plaque Agarose | Biozym | 840101 | 1% w/v in H2O |
| Deoxyribonuclease I from bovine pancreas, 2000 Kunitz units/vial | Sigma-Aldrich | D4263 | freshly dissolve content of 1 vial in 300 μl DMEM |
| DMEM | Gibco | 22320-022 | used as provided by manufacturer (Low Glucose, Pyruvate, HEPES) |
| cell strainers (100 μm, 75 μm) | BD Falcon | 352360, 352350 | |
| nylon mesh(48 μm, 30 μm) | Bückmann GmbH | 03-48/26-1020, 03-30/18-108 | |
| CellTrics 50 μm filter | PARTEC | 04-0042-2317 | |
| anti-mouse CD16/CD32 | BioLegend | 101302 | clone 93; purified |
| anti-mouse F4/80 | BioLegend | 123116 | clone BM8; APC coupled |
| anti-mouse CD11b | BioLegend | 101208 | clone M1/70; PE coupled |
| anti-mouse CD11c | BioLegend | 117310 | clone N418; APC coupled |
| anti-mouse CD45 | BioLegend | 103102 | clone 30-F11; purified |
| anti-mouse CD19 | eBioscience | 12-0193-83 | eBio 1D3; PE coupled |
| polyclonal goat anti-rat IgG | BD Pharmingen | 550767 | polyclonal, PE coupled |
Antibodies coupled to alternative fluorochromes can be used, depending on the flow cytometer and lasers available. |
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