作者想对她的帮助与细胞分选和彼得莫特拉姆在路易斯安那州立大学流式细胞仪核心设施，协助他与显微感谢玛丽莲迪特里希。这项工作是由乘务员医学研究所，路易斯安那州立大学的竞争的研究计划奖，以及美国国立卫生研究院/ NIAID的资助P20 RR020159和R03AI081171。

1。肺灌注和单细胞悬液<ol><li>安乐死鼠标，通过股静脉与腹腔注射氯胺酮/甲苯​​噻嗪麻醉剂的混合物（氯胺酮1.8毫克/鼠标，甲苯噻嗪0.19）和放血</li><li>切割和腹膜外的皮肤轻轻地拉了回来，暴露胸腔。继续打开切割肋骨暴露的心脏和肺的隔膜。 </li><li>轻轻灌注的肺，用5 ml注射器充满EDTA的HBSS（1MM EDTA钙/镁免费的HBSS）。连接25个G的针头和针插入右心室。为了防止在注射过程中的泄漏，安全使用镊子，针周围的心肌组织。轻轻注入的解决方案，同时保持恒定的压力。准确的灌注会导致肺胀，粉红/白颜色的变化。 </li><li>删除淋巴结放弃任何从这个组织的潜在污染。 </li><li>收集肺组织，在冰上转移到培养皿，并斩小块，用刀片。 </li><li>转移到一个gentleMACS彗星含有胶原酶消化溶液5毫升/肺管接地组织（胶原酶1A型为0.5 mg / ml加IV型牛胰脱氧核糖核酸酶20微克/毫升含5％胎牛血清，100 U / ml青霉素和100μg的HBSS / ml链霉素）。 </li><li>使用gentleMACS dissociator获得单细胞悬液。选择方案：m_lung_01。在37 ° C孵育30分钟的样品。摇管每隔5分钟重新悬浮组织碎片。继续执行程序m_lung_02。 </li><li> 10分钟335 X克的样品转移到15毫升锥形管和离心4 ° C 。这一步后，所有的试剂和离心保持在4 ° C，以防止细胞活力下降。 </li><li>弃上清，加入2毫升的ACK裂解在室温下1分钟的缓冲裂解其余的红血细胞。 13细胞与洗毫升的冷PBS/0.5％BSA和离心10分钟335 X克在4 ° C。 </li><li>倒掉上清，重悬细胞总数在5毫升冷PBS/0.5％BSA和通彻100微米尼龙网。 </li><li>确定使用台盼蓝拒染细胞总数。 </li></ol> 2。磁隔离和表面CD11c +细胞的富集<ol><li>在4 ° C离心​​10分钟200 ×克单细胞悬液，弃上清。 </li><li>重悬细胞沉淀在400分离缓冲液（PBS，0.5％BSA和2毫米EDTA）每10共8个细胞。块FC -介导的非特异性抗体结合anti-CD16/CD32抗体（0.5μg/ 10 6细胞）使用30分钟，在4 ° C。 </li><li> 10分钟200 ×克洗净细胞10毫升冷分离缓冲和离心机在4 ° C。 400分离缓冲液重悬细胞。 </li><li>加入100μL微珠表面CD11c每10 8 </SUP&gt;细胞总数。拌匀，孵育15分钟在冰箱（2-8℃）。 </li><li>洗细胞在4 ° C 10分钟200 ×克10毫升冷分离缓冲和离心，弃上清。 </li><li>在3毫升冷分离缓冲液重悬细胞沉淀。 </li><li>执行使用选择的程序posseld autoMACS临TM分离磁选。收集了积极的一小部分（0.5毫升）。 </li></ol> 3。常规树突状细胞（CDC）隔离<ol><li>孵育丰富的CD11c阳性细胞悬液与反表面CD11c PE - Cy7和anti-IA/IE（MHC -Ⅱ）- FITC抗体为30分钟，在4 ° C。优化的抗体浓度为0.5微克/ 10 6细胞。 </li><li>用冷PBS/0.5％BSA和离心10分钟335 X克在4 ° C的细胞PBS/0.5％BSA 0.5毫升悬浮细胞，并通过一个40微米尼龙网。 </li><li>继续进行排序细胞，使用流式细胞仪唱腔细胞分选纯度模式。添加100μLPBS/0.5％BSA的收集管，以防止纯化细胞的损害。整个细胞分选过程中的冷藏温度保持在你的细胞。 </li></ol> 4。获得单细胞悬液和表面CD11c +细胞富集的替代协议<ol><li>更换gentleMACS dissociator，从步骤1.5接地的肺组织可转移至15 ml锥形管的胶原酶每肺癌和孵化溶液5毫升含1小时在37 ° C涡细胞每隔15分钟，以重新悬浮组织碎片。组织扰乱通过样品和通过连接到20 G针3毫升注射器的6-8倍。避免在这个过程中的气泡，否则将受到损害细胞的活力。一旦获得单细胞悬液，继续加强1.8。 </li><li>在更换AutoMACSPro 商标分离表面CD11c细胞富集的磁性隔离器，也可以通过两个MAC列的细胞悬液，手工进行。样本的大小而定，选择适当的列会有所不同。 </li></ol> 5。代表性的成果： 龙CDC确定的CD11c 您好 / MHC -Ⅱ 您好细胞群。如图。 1，疾病预防控制中心代表所占比例较小为1.4％时相比，其他一些组织，如脾（4.8％）。然而，在对比脾，肺的CD11c阳性细胞表达MHC -Ⅱ的不同金额，包括比CDC不同的细胞群。单细胞的准备工作后，总肺细胞的细胞产量约3.0〜3.8 × 10 7细胞/肺癌的60-70％，从一个小超过1％的可行性代表CDC的一个子集。这个百分比增加后表面CD11c的磁隔离肺总量CDC是递增约10吨IMES（&gt; 16％）从原来的准备（图2A）。然而，表面CD11c细胞表达MHC -Ⅱ（ 嗨的CD11c / 罗 MHC -Ⅱ，巨噬细胞）的低量，高污染的细胞群（&gt; 70％）与肺部疾病预防控制中心的一个子集混合。如图。 2B，这些细胞被淘汰后，CDC纯度达到96％时，细胞分选步骤。疾病预防控制中心后，整个过程通常产量为5 × 10 4细胞/肺。显微表明中华网与典型的树突（上图）四舍五入细胞的形态特征。另一方面，表面CD11c 您好 / 罗 MHC -Ⅱ代表了形态不同的子集，显示细胞突起，这是典型的巨噬细胞（巨噬细胞， 下半部分）12。 <img alt="图1" src="/files/ftp_upload/3563/3563fig1.jpg" /> 图1。差分直流变频在小鼠肺和脾脏 。龙一个ND C57BL / 6小鼠的脾组织收集和处理与胶原酶。胶原酶消化，抗鼠的CD11c - PE - Cy7和抗小鼠IA / IE - FITC细胞染色。代表流式细胞仪图显示在肺，脾（CDC百分比的CD11c 您好 / MHC -Ⅱ 您好 ）。 <img alt="图2" src="/files/ftp_upload/3563/3563fig2.jpg" /> 小鼠肺单细胞悬液与反表面CD11c磁珠标记，并通过自动细胞分离A）代表图显示CDC的百分比（嗨的CD11c /您好MHC -Ⅱ） 和图2。小鼠肺疾病预防控制中心隔离 。 “巨噬细胞（巨噬细胞表面CD11c，您好/罗MHC -Ⅱ）表面CD11c富集后的种群。进一步丰富分数染色其次，使用抗鼠的CD11c - PE - Cy7和抗小鼠IA / IE - FITC。双阳性细胞进行分类，并通过流式细胞仪分析。要确定它们的形态，细胞cytospun和修改后的Wright - Giemsa染色染色B）代表图显示CDC的百分比，巨噬细胞和细胞分选后。显微显示肺疾病预防控制中心和肺巨噬细胞的形象代表。比例尺= 20微米。

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作者什么都没有透露。

肺鼠标直流隔离的呼吸道刺激广泛的研究的一个重要的技术。取得这些细胞的过程中，包括防止损失的细胞以及细胞活力和纯度的关键步骤。肺灌流前收集将有助于消除任何周围细胞以及减少污染物的红细胞。使用自动分解，可当处理大量的肺部advanteogus，否则，你可以选择的替代协议，牢记分散胶原酶消化后的组织应该做的，轻轻的时候手动完成。胶原酶溶液中的肺组织的潜伏期，而不应超出?...

<table width="200" border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> ACK裂解缓冲</td><td> Invitrogen公司</td><td> D6 - 0005DG </td><td></td></tr><tr><td>抗鼠的CD11c（HL3） </td><td> BD Pharmingen公司</td><td> 5580979 </td><td> PE - Cy7共轭</td></tr><tr><td>抗小鼠IA / IE（269） </td><td> BD - Pharmingen公司</td><td> 553623 </td><td> FITC标记</td></tr><tr><td> Collangenase类型1A </td><td>西格玛</td><td> 9891 - 500MG </td><td></td></tr><tr><td>细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340，352360 </td><td></td></tr><tr><td>表面CD11c（N418）微球</td><td> Miltenyi </td><td> 130-052-001 </td><td></td></tr><tr><td> DNA酶我</td><td>西格玛</td><td> D5025 - 150KU </td><td></td></tr><tr><td>汉克的平衡盐溶液</td><td> Invitrogen公司</td><td> 14170 </td><td></td></tr><tr><td> HEPES缓冲溶液</td><td> Invitrogen公司</td><td> 15630 </td><td></td></tr><tr><td>培养皿60毫米</td><td>屋宇署猎鹰</td><td> 351016 </td><td></td></tr><tr><td> GentleMACS™彗星管</td><td> Miltenyi </td><td> 130-093-237 </td><td></td></tr><tr><td> dissociator温和MACS </td><td> Miltentyi </td><td> 130-093-235 </td><td></td></tr><tr><td> AutoMACS，临™ </td><td> Miltenyi </td><td> 130-092-545 </td><td></td></tr><tr><td> FASCS咏叹调</td><td>屋宇署</td><td></td><td></td></tr></table>

isolation of mouse lung dendritic cells

龙树突状细胞（DC）感应入侵的病原体1,2以及耐受性反应的控制呼吸道发挥基础性作用。肺癌树突状细胞至少有三个主要的子集已在小鼠描述：传统的直流（CDC）4，浆DC（PDC）和生产干扰素的杀手DC（IKDC） 6,7。 CDC的一个子集，是最突出的DC在肺癌 8的子集。 已知识别DC亚群的共同标记的CD11c，I型跨膜整合（β2），也是在单核细胞，巨噬细胞，中性粒细胞和一些B细胞9表达。在一些组织中，作为一个标记，以识别鼠标DC表面CD11c是有效的，在脾，其中大部分表面CD11c +细胞代表CDC的子集，它表达的主要组织相容性复合体Ⅱ类（MHC -Ⅱ ）的高层次。然而，肺组织的地方是一个更异构端DC亚群，是一个不同的细胞群的比例较高，表示回合MHC -Ⅱ水平低的CD11c高水平。的CD11c 您好 MHC -Ⅱ 老龙的细胞群，根据其特性和大多是其表达F4/80，脾巨噬细胞标记，已被确定为10日和最近肺巨噬细胞，作为一个潜在的DC 前体11。 相比之下鼠标PDC，在肺部的免疫反应的疾病预防控制中心的具体作用的研究一直局限于由于缺乏一个特定的标记，在这些细胞的分离，可以帮助。因此，在这项工作中，我们描述了一个过程来分离高纯度的小鼠肺疾病预防控制中心。隔离肺的DC亚群代表一个非常有用的工具，以获得深入了解这些细胞的功能，在应对呼吸道病原体以及环境因素可以触发主机在肺部的免疫反应。

Watch this Scientific Journal Video about Isolation of Mouse Lung Dendritic Cells at JoVE.com

Isolation of Mouse Lung Dendritic Cells

描述一个高度纯化的小鼠肺树突状细胞的准备。具体的重点是给传统的树突状细胞亚群的隔离。

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 ...

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JoVE Journal

Immunology and Infection

28.9K 次观看.  Louisiana State University. 描述一个高度纯化的小鼠肺树突状细胞的准备。具体的重点是给传统的树突状细胞亚群的隔离。

视频: Isolation of Mouse Lung Dendritic Cells

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

小鼠腹膜腔细胞的分离

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

科研

免疫与感染

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

小鼠树突状细胞亚群的分离的高效和高产的方法

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...

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

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 quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

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 ...

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 ...