作者承认，从德意志研究联合会SI-1281/4-1的和BIOSS从弗赖堡大学医学系（CS）的补助金。

1。致病抗体的制备注意：该抗体的纯化和浓度应在同一天进行，作为抗体是不是要被存储在0.1M甘氨酸pH为2.5-3（洗脱缓冲液）过夜（ON）。 亲和纯化的IgG：使用免疫兔血清25毫升： <ol><li>解冻后的兔血清对在4°C，混合，以1:1 1xPBS（运行缓冲液），并在1260 XG离心10分钟，在20°C。 ，用滤纸过滤步骤可能包括离心后的情况下，血清脂肪。 </li><li>同时，洗G矩阵填充柱，10层的体积（床体积=矩阵体积）的1XPBS（缓冲区）的蛋白质。 </li><li>应用稀释并过滤血清列，并在室温（RT）的摆动平台上孵育1小时。 </li><li>收集流过（FT），在50毫升的小瓶管。不要丢弃它，直到浓度和纯化的IgG滴度的是已知的。 </li><li>洗净，用5柱床体积的1XPBS矩阵，并在此期间收集洗脱的IgG级分的制备50毫升Falcon管中。移液器1毫升pH值中和缓冲液：1 M TRIS每个管（否则1:20 TRIS缓冲液的比例，相对于要收集的洗脱液的量）中的pH值为10。 </li><li> 100-150毫升洗脱缓冲液：0.1 M甘氨酸pH值2.5-3列，并收集50毫升组分（S）。翻转管2到3倍，测量pH值和添加中和缓冲区，直到你已经达到了pH值7.2-7.4。 </li><li>收藏报错几滴的IgG的洗脱液，使用洗脱缓冲液作为标准具测量在280nm处的OD值。如果OD &lt;0.1停止收集。 </li><li>重新生成的蛋白质G矩阵，并按照制造商的说明将其存储。 </li></ol>通过超滤用Amicon管浓度： <ol><li>通过离心15-20分钟，洗净的Amicon Ultra（15毫升/ 30KDa）超滤管1XPBS在3220 XG在4°C。丢弃从AMICON FT。 </li><li>填充Amicons为25-30分钟，在3,220 xg离心和4℃下用15毫升的洗脱IgG级分和离心机，离心时间总是取决于洗脱液中的蛋白质含量。 </li><li>丢弃FT和重复的超滤，直到你有没有洗脱馏分离开。 </li><li>清洗浓缩抗体广泛的两倍，：1XPBS 25-30分钟离心。 </li><li>收集的“粘性”，淡黄抗体溶液在最终体积为1,500-2,000微升1XPBS从的Amicon过滤器。 </li><li>测量浓度的IgG制剂，使用分光光度计或的NanoDrop： 浓度。 （毫克/毫升）= A（280纳米）×稀释倍数/ 1,4 </li><li>洗净，按照制造商的说明存储Amicons。 </li></ol> 2。注射到小鼠体内的胶原纤维VII的特异性IgG 在实际的实验过程开始之前，确保实验的亲，tocol的书面和所有材料都准备好了实验。 <ol><li>准备的数据表疾病的得分，ELISA试剂盒，免疫荧光分析（IF）和髓过氧化物酶（MPO）检测。 </li><li>标记管的血液和器官，的cryomolds以及组织学处理和嵌入磁带。 </li><li>抗体溶液应准备新鲜或解冻的股票和特点，考虑到他们的反应（滴度IF显微镜和/或ELISA）。无菌过滤抗体溶液应是稀释在PBS中，在一个方式，所需的注射剂量为250-1000微升之间变化。请确保您有足够的抗体来完成整个实验。 </li><li>准备新鲜抗凝血剂肝素20 U / ml和8.7毫克/毫升氯胺酮/ 1.3毫克/毫升甲苯噻嗪麻醉混合物。当牺牲的老鼠标记的试管中，用3.7％甲醛准备。 </li><li>消毒手术器械（手术刀，剪刀，细一点，CURV编辑forcipes），并准备注射器（胰岛素注射器，1和2毫升注射器），针，数码相机和额外的记忆卡。 </li></ol> 注：执行所有的程序，氯胺酮/甲苯噻嗪或异氟烷narcotized的小鼠。 异氟醚选项的日常皮肤检查是首选的麻醉老鼠一样，迅速 ​​恢复。然而，在拍照时，87 mg / kg氯胺酮和13毫克/公斤甲苯噻嗪通常是皮下注射。对于安乐死，氯胺酮/甲苯​​噻嗪剂量增加至130 mg / kg氯胺酮和20毫克/公斤甲苯噻嗪。 <ol><li>为自己的整体健康状况，皮肤和毛皮的外观检查已标记的动物，称重，并测量他们的耳朵厚度（始终坚持同侧耳）。 </li><li>注册在临床评价表的观测值和变化。 </li><li>含有相同体积的抗凝血剂中的注射器从尾静脉收集20-30微升（2-3滴）血液。 </li><li>消毒皮毛和皮肤用80％的乙醇。 </li><li>抗体溶液注入背部皮下使用胰岛素注射器。（对于某些网站（如耳），只有少量（10〜50微升）是可行的注入。） </li></ol>注射剂，应每隔一天重复的4倍。 BALB / c和C57BL6小鼠的体重约20克开始患上这种疾病后3-4天的第一届政府的400到750微克/克体重/注射自体抗体。 当完成实验： <ol><li>将小鼠注射130 mg / kg和20 mg / kg的氯胺酮/甲苯​​噻嗪的皮下深麻醉下，从后腔静脉抽血他们和切心。 </li><li>收集如皮肤：尾巴，耳朵，血液和食道的器官/组织样本，并把它们标记和PBS或3.7％甲醛填充管。 </li></ol>当返回到实验室检查： <ol><li>离心1200×g离心10分钟，在室温下分离血浆的血液，转让等离子的正确标记的试管中，并将其储存。 </li><li>嵌入皮肤活检的最佳切割温度介质使用cryomolds，适当的标签，并将其储存在-80°C。 </li><li>将意味着在组织学标记嵌入模具和他们保持在3.7％的甲醛，直到石蜡包埋组织样本。 </li></ol> 3。临床评价疾病的严重程度小鼠应每天检查皮肤和粘膜的病变，应使用适当的形式记录调查结果。早安乐死的标准是皮肤的病变影响，20％的体表和连续三天的身体总重量的5％至10％的重量损失，计数的疾病5分（ 表1）。 <ol><li>把鼠标放在它的肚子。开始的头部区域，即用正确的耳朵和检查水疱，糜烂，以及地壳内的外侧。继续以同样的方式与左耳。 </li><li>左眼和右眼被控制的迹象，红斑，脱发，糜烂和/或地壳。 </li><li>刷到明年，额头和吻部地区持续的鼻子，嘴唇和口粘膜的腹侧部分。 </li><li>检查右前腿的爪子开始，向上移动，切换到左前腿。的右和左后腿的相同的程序。 </li><li>刷机通过弯曲的细点镊子颈部和背部面积的皮毛上。 </li><li>转动鼠标在它的后面，并检查的腹侧部分以及腹侧四肢如前面相同的方式。 </li><li>检查的尾巴。 </li><li>拍照的有关身体部位的小鼠疾病。必须注意的图片的质量和数量。 </li><li>计算疾病的临床得分。 </li></ol> 4。皮肤和血浆样品的分析<ol><li>准备，存储和/或处理所有收集到的组织，根据该计划。 </li><li>切冷冻组织切片检测兔抗体和小鼠补体系统的组成部分。 </li><li>分析不同的冰冻组织提取物和/或器官的蛋白质和酶的含量（MPO测定）。 </li><li>第石蜡包埋组织，并与H＆E染色的可视化组织损伤的程度。 </li><li> ELISA，免疫印迹分析血浆。 </li></ol>

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没有利益冲突的声明。

被动转移到实验动物的自身抗体是一个重要的方法展示他们的致病性，-12。通过这种方法获得的动物模型，除了是自身免疫13的间接证据，允许传出的调查阶段的致病机理。抗体诱导的粒细胞依赖的皮肤起泡性大疱性表皮松解症acquisita（EBA）的被动转移模型被用于解剖组织损伤的机制，在自身免疫性皮肤病。它的出现，作为一个精美的启发性疾病模型研究的根本，远远超出对胶原?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>重组抗原</td><td></td><td></td><td>的质粒编码的重组形式的小鼠的胶原VII可从相应的作者。 </td></tr><tr><td>免疫兔血清</td><td> <a href="http://www.eurogentec.com/" target="_blank">www.eurogentec.com</a> </td><td></td><td>我们有免疫200μg的抗原，在2周的时间间隔的3倍的新西兰白兔。为了这个目的，我们已经使用了服务Eurogentec SA，比利时。 </td></tr><tr><td> G蛋白琼脂糖</td><td>罗氏应用科学部</td><td> 11243233001 </td><td></td></tr><tr><td> Balb / c小鼠</td><td> Charles River实验室</td><td></td><td></TD&gt; </tr><tr><td> OCT化合物，组织康</td><td>樱花Finetek </td><td> 4583 </td><td> OCT，最佳切削温度</td></tr><tr><td> Cryomold标准</td><td>樱花Finetek </td><td> 4557 </td><td> 25毫米×20毫米×5毫米</td></tr><tr><td> Cryomold中间</td><td>樱花Finetek </td><td> 4566 </td><td> 15毫米×15毫米×5毫米</td></tr><tr><td>统一链接Einbettkassette的</td><td> R. Langenbrinck </td><td> 09-0503 </td><td>组织学处理和嵌入箱</td></tr><tr><td> SPEZIAL-Tatowierfarbe施瓦茨的</td><td> H. Hauptnerund和理查德Herberholz的GMBH＆CO KG </td><td> 71492000 </td><td>纹身贴</td></tr><tr><td> Tatowierzange TZ1 </td><td> EBECO E.贝克尔＆CO GMBH </td><td></td><td>纹身设备</td></tr><tr><td>肝素</td><td>卡尔·罗斯GMBH＆CO。 </td><td> 7692.1 </td><td></td></tr><tr><td>甲醛37％ </td><td>卡尔·罗斯GMBH＆CO。 </td><td> 7386 </td><td></td></tr><tr><td>盐酸氯胺酮</td><td> Sigma-Aldrich公司Chemie GmbH公司</td><td> K2753-1G </td><td></td></tr><tr><td>赛拉嗪盐酸盐</td><td> Sigma-Aldrich公司Chemie GmbH公司</td><td> X1251-1G </td><td></td></tr><tr><td>无菌PBS </td><td> Biochrom </td><td> L182-50 </td><td></td></tr><tr><td>数码相机</td><td>尼康</td><td> COOLPIX 5400数码相机</td><td></td></tr><tr><td>注射器驱动0.45微米过滤装置</td><td>密理博</td><td></td><td></td></tr><tr><td>卡尺</td><td>三丰7309 </td><td> 1667338 </td><td>派睿电子（经销商） </td></tr><tr><td>疾病计分表</td><td></td><td></td><td>例如机箱。中美战略经济对话</td></tr></tbody></table>

granulocyte-dependent autoantibody-induced skin blistering

自身免疫性现象发生在健康人，但，自我宽容失败时，可能会导致自体免疫反应在具体的病理。据Witebsky的假设，诊断为自身免疫性疾病中的标准之一是在实验动物中的疾病的再现由被动转移的自身抗体。大疱性表皮松解症acquisita（EBA），原型器官特异性自身免疫性疾病，皮肤和粘膜，一些实验模型，最近成立的。在动物模型中描述在我们目前的工作中，纯化的IgG抗体对拉伸的200个氨基酸（氨基酸757-967）的胶原纤维VII反复被注入到小鼠体内，再现起泡表型以及直方和免疫病理学特征对人体EBA 1。全面爆发普遍的疾病，通常第一次注射后5-6天，疾病的程度与剂量的管理Çollagen的VII-特异性IgG。在实验EBA组织损伤（水疱形成）根据粒细胞的募集和活化组织结合自身抗体-2，-4。因此，该模型允许粒细胞依赖的炎性途径参与自身免疫性组织损伤的解剖，作为模型只再现传出神经的自体免疫反应的T细胞独立的相位。此外，它的值被强调的一些研究表明自身抗体在体内的泡罩诱发电位和调查-6 EBA 1,3，水疱形成的机制。最后，这种模式将极大地促进新的自身抗体引起的疾病的消炎疗法的发展。总体而言，EBA被动转移动物模型的可访问性和指导性的疾病模型，将有助于研究人员不仅分析的的EBA发病，但回答基本的生物学和临床重要的自身免疫问题。

的被动转移抗原特异性抗体，结果在一个完全成熟的病鼠在临床，病理和免疫病理的水平，类似的人EBA。水疱，痂皮，糜烂和脱发发展的耳朵，猪嘴，爪，腿，背部和眼睛周围的老鼠。第一临床疾病的征兆，很可能会出现在耳朵上和/或头部区域。如果在中冰冻切片的病灶周围皮肤的真皮表皮交界处，检测到特异性的兔IgG和鼠补体C3沉积直接。在皮损部位表皮下疱和炎症浸润的组织学。 <p class="j...

Watch this Scientific Journal Video about Granulocyte-dependent Autoantibody-induced Skin Blistering at JoVE.com

Granulocyte-dependent Autoantibody-induced Skin Blistering

在动物模型中描述在我们目前的工作中，纯化的IgG抗体对拉伸的200个氨基酸（氨基酸757-967）的胶原纤维VII反复被注入到小鼠体内，再现起泡表型以及直方和免疫病理学特征对人体acquisita大疱性表皮松解症（EBA）<sup&gt; 1</sup&gt;。

粒细胞相关的自身抗体引起的皮肤起泡

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

granulocyte-dependent-autoantibody-induced-skin-blistering

Research

JoVE Journal

Immunology and Infection

10.4K 次观看.  University of Freiburg. 在动物模型中描述在我们目前的工作中，纯化的IgG抗体对拉伸的200个氨基酸（氨基酸757-967）的胶原纤维VII反复被注入到小鼠体内，再现起泡表型以及直方和免疫病理学特征对人体acquisita大疱性表皮松解症（EBA） 1。

视频: Granulocyte-dependent Autoantibody-induced Skin Blistering

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

科研

免疫与感染

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

单细胞悬液的细胞荧光分析从不同的鼠标皮肤区域准备

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em&gt;疟原虫</em&gt;孢子

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

鼠全层皮肤移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

通过“Acufection”和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...