这项工作是由CIHR和CF加拿大赠款博士吉姆胡锦涛和加拿大的CF拉胡尔Kushwah授予博士学位奖学金。

1。致敏的小鼠卵白蛋白<ol><li>准备解决的OVA（V级;西格玛，MO）在无菌PBS在1毫克/毫升的浓度（溶液可存放于-80°C）。 </li><li>为了准备卵明矾的混合物，在管的明矾和卵清蛋白溶液中滴加的方式加入，而在1:1的比例涡旋管。混合搅拌30分钟，混合后马上使用。 </li><li>用1毫升注射器，0.2毫升的混合物注入小鼠腹腔注射2周后再次重复。 </li></ol> 2。 FITC标记的卵清蛋白与小鼠鼻内挑战<ol><li>卵明矾第二次注射后7天内，老鼠是准备接受挑战OVA-FITC标记滴鼻。 </li><li>准备的卵-FITC（Sigma公司）的解决方案，使用浓度为1毫克/毫升，分装店在无菌PBS -80°C。 </li><li>无菌纱布放置在50毫升猎鹰管的底部，并在化学ĤOOD，添加到纱布5毫升Aerrane。为了麻醉小鼠，直接进入管约5-10秒的动物。 </li><li>在直立位置，并使用无菌枪头，吸管100微升到老鼠的鼻孔OVA-FITC标记在下拉明智的时尚解决方案，保持麻醉的动物。 </li><li>重复鼻内挑战与未来两天的OVA-FITC标记。 </li></ol> 3。从肺部及引流淋巴结的单细胞悬液的制备<ol><li>使用腹腔注射Euthanyl的牺牲小鼠。 </li><li>为了减少巨噬细胞的污染，其中直流分析复杂，进行肺灌洗。到肺灌洗，简要地暴露小鼠气管插管导管和注射器，用5毫米EDTA冷PBS，下呼吸道冲洗3次，以去除细胞从肺泡空间。 </li><li>灌注10毫升的PBS含10 U / ml的剖检可见肺部在通过心脏的右心室，以消除肺血管中的血细胞。执行灌注，直到肺部变白，去除大部分血细胞的指标。消除污染外周血细胞，这一点尤为重要。 </li><li>解剖肺部和删除纵隔淋巴结（MLN），并将其放置在一个单独的盘的淋巴结。 （尽量减少照射到组织，以防止FITC荧光猝灭。） </li><li>放在培养皿的肺部和百果用剪刀。下一步消化肺部在37°C使用250 U / ml的胶原酶D类（罗氏公司），并添加EDTA（10毫米终浓度）为接下来的5分钟停止胶原酶活性为25分钟</li><li>通过消化肺细胞过滤100微米的碎片和执行低渗溶解去除红细胞。单细胞悬液，准备区议会进一步分析。 </li><li>为了准备从单细胞悬液，肠系膜淋巴结，T缓解使用胶原酶细针和消​​化，如上所述，随后通过一个细胞过滤紧张MLN的。 </li></ol> 4。直流染色标记，以评估成熟/迁移<ol><li>为了确定在肺部的DC，染色CD11c和CD11b的表现。此外，以评估为成熟，染色CD86和CD80上调，这是作为区议会接受成熟的表现。素CD11c +细胞CD11b + OVA-FITC标记+在MLN的细胞被确认为肺区议会迁移从肺部的响应卵呼吸道挑战的MLN的。 </li><li>暂停浓度在10×10 6细胞/毫升，在流式细胞仪缓冲（PBS 1％FBS和1mM EDTA）和流式细胞仪染色的细胞悬液/管分装150微升的细胞。肺区议会的识别，需要以下的污渍：不染​​控制（不暴露于OVA-FITC标记的小鼠肺细胞），OVA-FITC标记的控制，CD11c的单一控制，CD11b的单一控制，CD11b的+ CD11c的+双重控制，细胞CD11b + OVA-FITC标记+双控，素CD11c + OVA-FITC标记+双控，MHC II类单一的控制，CD86的单一控制，CD80的单一控制和样品染色卵-FITC标记+素CD11c +细胞CD11b + MHCII +卵-FITC标记+ CD11c的细胞CD11b + CD86的+和卵-FITC标记+素CD11c +细胞CD11b + CD80 +。 </li><li>为了评估DC的迁移，执行从MLN的单细胞悬液绝对细胞计数，并随后准备以下管：不染控制（MLN的细胞不暴露于OVA-FITC标记的小鼠），OVA-FITC标记的控制，素CD11c +单一的控制，细胞CD11b +双重控制，细胞CD11b + OVA-FITC标记+双控制，素CD11c + OVA-FITC标记+双控制和素CD11c +细胞CD11b + OVA-FITC标记+样本。 </li></ol> 5。代表结果腹腔致敏诱发呼吸道过敏性炎症的时间点很重要，应进行如图1所示。小鼠腹腔致敏和呼吸道卵的挑战后，确认诱导的气道过敏性炎症，有的可以牺牲和组织学分析，可进行肺段， 如图2所示。炎性细胞的存在可以证实苏木精和曙红染色（ 图2A）和粘液生产的存在，可以通过定期酸希夫染色（ 图2B）进行评估。总之，这将确认后诱导的气道过敏性炎症8 OVA致敏小鼠卵挑战。相比之下，生理盐水治疗小鼠的肺部分预计将随着没有任何粘液生产无任何炎症。此外，以下卵的挑战，肺区议会unde<html>的RGO成熟和随后迁移到淋巴结。预计的CD11c +细胞在小鼠以OVA致敏和挑战，从纵隔淋巴结（MLN）进行分析，显示出较高的CD11c +细胞的比例相比，生理盐水治疗的老鼠（ 图3A）。此外，+ OVA-FITC标记+ OVA致敏和小鼠肠系膜淋巴结细胞的CD11c + CD11b的绝对细胞计数分析，预计显示显着升高（即高出数倍）计数，生理盐水治疗小鼠（同行图3B）。 <img alt="图1" src="/files/ftp_upload/4014/4014fig1.jpg" /> 图1。诱导卵白蛋白（OVA）诱导小鼠过敏性气道炎症，随着呼吸道挑战与FITC标记的卵清蛋白（OVA-FITC标记）的实验协议。 fig2.jpg“/&gt; 图2。致敏卵呼吸道挑战导致呼吸道过敏性炎症，苏木精和曙红染色显示肺段（一）确定感应，也导致呼吸道粘液，碘酸希夫肺的染色确定的生产（二）所示的部分。 <img alt="图3" src="/files/ftp_upload/4014/4014fig3.jpg" /> 图3。OVA-FITC标记的挑战诱导DC的迁移从肺部纵隔淋巴结（MLN）。 （一）流式细胞仪图描绘在控制或OVA致敏小鼠肠系膜淋巴结的CD11c +细胞的比例。 （二）素CD11c +细胞CD11b + OVA-FITC标记的生理盐水或OVA致敏小鼠肠系膜淋巴结+细胞绝对计数。 * P &lt;0.05。 <a href="http://www.jove.com/files/ftp_upload/4014/4014fig3large.jpg" target="_blank">点击这里查看大图</a> 。

<ol>
	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, 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=Dendritic+cells+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

没有利益冲突的声明。

这里介绍的方法提供了一个流式细胞仪分析肺区议会，上呼吸道挑战OVA-FITC标记的交付为基础的基础的做法。这允许选择性的肺区议会，占用OVA-FITC标记，因此直流人口，这是监测在OVA诱发的过敏性气道炎症过程，在呼吸道的免疫反应参与的有效的监测。在对照组，在过敏性气道炎症的情况下，的洄游区议会号码（卵-FITC标记+区议会）的肠系膜淋巴结中确定的指示直流迁移，基础利率而过敏性气道...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>卵清蛋白</td><td> Sigma-Aldrich公司</td><td> A5503 </td></tr><tr><td>明矾</td><td> Thermo Scientific的</td><td> 77161 </td></tr><tr><td> OVA-FITC标记</td><td> Sigma-Aldrich公司</td><td> A9771 </td></tr><tr><td>胶原酶ð </td><td>罗氏公司</td><td> 11088858001 </td></tr><tr><td>抗体</td><td> E-生物科学</td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

树突状细胞（DC）的启动适应性免疫应答的抗原特异性T细胞激活的关键球员。区议会是目前在稳定状态下的外围组织，区议会，然而，在抗原刺激的反应，采取抗原，并迅速转移到淋巴结，他们开始对T细胞反应的抗原1,2。此外，区议会也起到了关键作用，在启动自身免疫性以及过敏免疫反应3。 区议会发挥了至关重要的作用，同时启动免疫反应和诱导耐受性肺环境设置4。肺癌环境主要是耐受性，由于置身于广大环境抗原5阵列。然而，在一些人有一个宽容的突破，从而导致过敏和哮喘的感应。在这项研究中，我们描述了一个战略，它可以用来监测气道DC成熟和米格在抗原致敏使用的口粮。气道DC成熟和迁移的测量，允许在气道过敏性炎症免疫反应动力学的评估，并协助随后沿与免疫反应的基本机制的了解程度。 我们的战略是基于对卵清蛋白作为增敏剂的使用。卵清蛋白诱发过敏性哮喘是一种广泛使用的模型，以重现气道嗜酸性粒细胞浸润，肺炎和升高的IgE水平在哮喘6,7。致敏后，小鼠滴鼻交付FITC标记的卵，它允许特定的标签气区议会其中吸收卵的挑战。接下来，使用多个DC特异性标志物，我们可以评估这些区议会的成熟，并采用流式细胞仪还可以评估其迁移到淋巴结。

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

我们描述了一个战略，以监测肺部树突状细胞的成熟和迁移的卵在卵蛋白引起的过敏性气道炎症的设置。这种策略可以进行修改，以评估肺部树突状细胞的迁移，在感染的设置。

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

analysis-pulmonary-dendritic-cell-maturation-migration-during

Research

JoVE Journal

Immunology and Infection

14.9K 次观看.  McMaster University, Hamilton. 我们描述了一个战略，以监测肺部树突状细胞的成熟和迁移的卵在卵蛋白引起的过敏性气道炎症的设置。这种策略可以进行修改，以评估肺部树突状细胞的迁移，在感染的设置。

视频: Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

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 ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

在体内成像方法区分急性和慢性炎症

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

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 ...

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 ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...