Name: detection and isolation of viable mouse il-17-secreting t cells
Uploaded: 2008-12-18T00:00:00.000+00:00
Duration: 12 min 38 s
Description: Watch this Scientific Journal Video about Detection and Isolation of Viable Mouse IL-17-Secreting T Cells at JoVE.com

准备试剂<ol><li>含BSA（0.5％），和2毫米EDTA PBS缓冲区。由于气泡可以阻止磁珠分离柱，缓冲区，并储存于2-8 ° C，使用前需要脱气。 </li><li>我们用RPMI 1640培养基中含有5％的小鼠血清。培养液中不应该包含BSA或FCS，这些化合物会改变细胞刺激的特异性。 </li><li>小鼠IL - 17分泌含量 - 细胞富集和从美天旎生物技术检测试剂盒。该套件包含以下几部分组成的IL - 17的捕捉试剂，IL - 17检测抗体（生物素），抗生物素- PE，和反PE微球。 </li></ol>刺激的脾细胞<ol><li>此协议是执行中存在的消极和积极的控制，如未刺激的脾细胞和T细胞的染液，。该协议是使用无菌技术进行。 </li><li>准备一个单细胞悬液，小鼠的脾细胞中分离使用gentleMACS™Dissociator。通过细胞计数，细胞浓度应预先确定。颗粒的细胞在室温下10分钟，在200 × g。 </li><li>离心后，吸出上清液用吸管关闭沉淀。不要调迁管，以避免损失颗粒。 </li><li>现在，重悬的细胞在培养液中，然后添加到良好。添加足够的介质浓度为每毫升1000万细胞和细胞每平方厘米500万。 </li><li>为了刺激免疫反应在悬浮细胞，我们添加离子霉素（1微克/毫升）和PMA（10毫微克/毫升）样品和混合解决方案，通过向上和向下轻轻吹打。然后井相应的标记。 </li><li>现在，我们将孵育3小时，我们的细胞，在37℃，没有搅拌，开始的刺激期。继续从刺激开始到IL - 17的分析，3小时，所以相应的计划。 </li><li>要停止分泌，细胞被放置在冰和我们收集的刺激细胞轻轻吹打冷缓冲区向上和向下。细胞，然后转移从井管，洗第二次。 </li><li>为了确保所有细胞都收集，它是一个好主意，在显微镜下检查你的菜。如果细胞仍然保持连接，您可以收集冲洗用冷水缓冲菜剩下的细胞。采用预分离过滤器，可去除细胞悬液中的任何细胞团块。 </li></ol>标签捕获试剂细胞<ol><li>这是最重要的要注意，这个实验工程优化，如果低于2％的IL - 17分泌细胞都存在。 </li><li>如果预计的IL - 17分泌细胞浓度大于2％，相应地调整卷。 <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="图8" width="500" height="162" /></li><li>此过程中的陷阱是一个潜在的交叉污染，渔获物试剂，它可以在标签发生时的双特异抗体非分泌性T细胞和陷阱从邻近的淋巴细胞分泌IL - 17的结合，从而产生误报。 </li><li>为了规避这个问题，关键是凉爽下来之前，标签和冷缓冲区的工作细胞缓慢的IL - 17的扩散和避免这种情况的交叉污染。此外，以维持细胞的冷，他们必须保持在一个定义的浓度。 </li><li>要开始标签的过程中，我们使用一百万个细胞，在15毫升的Closable管。如果要使用更高的手机号码，只需规模相应的所有卷。一旦已获得的最佳细胞浓度，加入10毫升冷缓冲洗细胞。 </li><li>离心10分钟在冷冻离心机（2-8℃）在300 × G细胞。 </li><li>离心后，完全用吸管吸出上清液。不要倒出上清液，因为这会导致细胞的损失和不准确的卷。重复的洗涤步骤，包括加入冷的缓冲液，离心和愿望10毫升。 </li><li>现在，我们有一个所需纯度的颗粒，在80μL冷培养基重悬细胞。给它们加上标签，现在我们将添加20μL小鼠IL - 17捕获试剂。 </li><li>在冰上5分钟的孵育细胞。 </li><li>在冰上5分钟的潜伏期后，取出管和稀释在10毫升37℃的温暖中期的细胞。然后安全MACSmix管肩管和管在37 ° 45分钟下连续运动的彗星。提高温度至37 ° C将重新启动细胞因子的分泌。 </li></ol>与IL - 17的检测抗体（生物素）和抗生物素PE标记的细胞<ol><li>经过45分钟的分泌期在37 ° C，立即在冰上放置管。这将阻止细胞因子的分泌。从这里就可以保持细胞在冰上的关键。与寒冷的缓冲区工作将避免交叉污染渔获物试剂。 </li><li>填充管冷缓冲区和离心机在300xg在2-8 ° C为10分钟。完全吸出上清液。重复清洗步骤有更多的时间。 </li><li>细胞沉淀悬浮于80μL冷缓冲管置于冰上。 </li><li>为了避免非特异性结合的抗体，我们建议除了加入10μlFCR阻断剂和5分钟的冰孵化。然后，我们添加20μL小鼠IL - 17检测抗体（生物素），结合生物素和10分钟在冰上孵育。 </li><li>再次，洗在300xg加入10毫升冷缓冲区和离心机在2-8 ° 10分钟之前我们已经做了的细胞。 </li><li>现在，吸出上清液，并在80冷缓冲液重悬细胞沉淀。 </li><li>加入我们的二级抗体，抗生物素- PE 20μL。 </li><li>细胞和二次抗体溶液混合，并再次孵育10分钟就冰管。 </li><li>一旦这第二个孵化完成后，用10毫升冷缓冲区和离心机的细胞。 </li><li>现在可以沉淀悬浮于500μL冷缓冲区。 </li><li>如果要作进一步的下游分析分离细胞，它们可以被磁标记与反PE的微珠和失散的手动或自动使用Mac列和MACS分离。 </li><li>现在，我们已经完成了脾的标签，并准备好进行分析。 </li></ol>流式细胞仪分析<ol><li>对于我们的分析，我们会比较刺激和未刺激的脾细胞，用流式细胞仪。 </li><li>之前，流式细胞仪分析，细胞染色为0.5微克/毫升与碘化丙啶门出死细胞。 MACSQuant™分析器装载每个样品20细胞，现在我们要看看样品中的相对抗体反应的散点图。稀有细胞分析的两个重要的考虑因素 - 如IL - 17阳性细胞 - 流式细胞仪检测： <ul><li>淋巴细胞上设置一个门，前向散射与侧散点图</li><li>出死细胞（碘化丙啶染色）和B细胞（这可能会导致非特异性背景染色）浇注，进一步提高检测的灵敏度</li></ul></li><li>我们用CD4 - APC抗体检测T细胞和CD45R/B220-PerCP抗体检测的B细胞。我们看到了样品的前部和侧散射性质和应用对淋巴细胞人口的门。 </li><li>第二个门被应用到染色的死细胞和彩色B细胞（Y轴）。这些细胞是T细胞的分析排除。 </li><li>在这个例子中，基本上是我们的阴性对照，我们可以看到，有很少IL - 17分泌的CD4阳性T细胞在未刺激样本 - 约0.003％（图4）。在刺激T细胞的人口来看，我们可以看到大量的CD4阳性T细胞分泌IL - 17 - 约0.367％（图5A）前分离磁选后MACS科技（图5B）和60.76％。 </li></ol>

所有的协议和数据表www.miltenyibiotec.com。 警告 试剂含有叠氮化钠。在酸性条件下的叠氮化钠产量hydrazoic酸，这是毒性极强。叠氮化合物，应与运行前丢弃的水稀释。这些预防措施建议，以避免在管道爆炸国情的发展可能的存款。

Th17细胞在适应性免疫和炎症反应中发挥不可或缺的作用，并在推动自身免疫性炎症的重要组成部分。 此视频文件，如何使用美天旎的鼠标IL - 17的分泌含量 - 细胞富集和检测试剂盒（PE）。执行此过程时，重要的是要在您的起始细胞制备的IL - 17分泌细胞的频率估计。在适当的细胞浓度的标签和细胞因子的分泌期防止其他细胞的交叉污染，在您的暂停，并确保可靠的结果。

<table><tbody><tr><td>Mouse IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) </td><td>Reagent</td><td>Miltenyi Biotec</td><td>130-094-213</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx&lt;/a&gt;</td></tr><tr><td>RPMI 1640</td><td>Medium</td><td>Miltenyi Biotec</td><td>130-091-440</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx&lt;/a&gt;</td></tr><tr><td>CD4-APC</td><td>Reagent</td><td>Miltenyi Biotec</td><td>130-091-611</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx&lt;/a&gt;</td></tr><tr><td>CD8a-FITC</td><td>Reagent</td><td>Miltenyi Biotec</td><td>130-091-605</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx&lt;/a&gt;</td></tr><tr><td>Propidium Iodide Solution</td><td>Reagent</td><td>Miltenyi Biotec</td><td>130-093-233</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx&lt;/a&gt;</td></tr><tr><td>FcR Blocking reagent, mouse </td><td>Reagent</td><td>Miltenyi Biotec</td><td>130-092-575</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx&lt;/a&gt;</td></tr><tr><td>Dead Cell Removal Kit </td><td>Reagent</td><td>Miltenyi Biotec</td><td>130-090-101</td><td></td></tr><tr><td>MACSmix Tube Rotator</td><td>Instrument</td><td>Miltenyi Biotec</td><td>130-090-753</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx&lt;/a&gt;</td></tr><tr><td>gentleMACS&amp;#8482; Dissociator</td><td>Instrument</td><td>Miltenyi Biotec</td><td>130-093-235</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx&lt;/a&gt;</td></tr><tr><td>autoMACS Pro Starting Kit</td><td>Instrument</td><td>Miltenyi Biotec</td><td>130-092-545</td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx&quot;&gt;http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx&lt;/a&gt;</td></tr><tr><td>MACS Columns</td><td>Tool</td><td>Miltenyi Biotec</td><td></td><td>&lt;a href=&quot;http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx&quot;&gt;http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx&lt;/a&gt;</td></tr><tr><td>MACS Separator</td><td>Tool</td><td>Miltenyi Biotec</td><td></td><td></td></tr></tbody></table>

detection and isolation of viable mouse il-17-secreting t cells

互助委员会的细胞因子的分泌检测技术，允许在可行的细胞因子分泌细胞的单细胞水平和敏感隔离分泌的细胞因子的检测。为了标签IL - 17分泌细胞，小鼠脾单细胞悬液，准备和刺激，在37 ° C至PMA /离子霉素诱导细胞因子的分泌。要停止分泌细胞，然后放置在冰暴露的IL - 17的捕获试剂，结合细胞表面的白细胞和IL - 17的CD45的分泌和细胞表面附近捕获，因为它是一个双特异抗体。分泌，然后重新开始提高温度至37 ° C和IL - 17是由Catch试剂被困。分泌，然后又停了下来，放置在冰细胞。要检测出被困IL - 17，第二个IL - 17特异性抗体结合，以生物素和抗生物素- PE抗体细胞培养。细胞现在可以直接通过流式细胞仪分析或准备与反PE标记的微球随后标签隔离和浓缩。

Watch this Scientific Journal Video about Detection and Isolation of Viable Mouse IL-17-Secreting T Cells at JoVE.com

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

此过程描述了小鼠Th17细胞的白细胞，积极分泌刺激后，IL - 17的检测和隔离。

可行的小鼠IL - 17分泌性T细胞的检测和隔离

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

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detection-and-isolation-of-viable-mouse-il-17-secreting-t-cells

Universidad San Sebastian

Research

JoVE Journal

Biology

11.2K 次观看.  Miltenyi Biotec,GmbH.  ChineseVersion JapaneseVersion 

视频: Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

科研

免疫与感染

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

IL-22表达淋巴细胞使用记者小鼠的可视化

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

破译急性 LCMV 感染中的早期滤泡辅助性 T 细胞定型术

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

作者聚焦:阐明急性 LCMV 挑战中 TFH 细胞分化的途径

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

可行的小鼠IL - 17分泌性T细胞的检测和隔离

副本

摘要

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此视频中的章节

隔离和幼稚的CD4 T细胞的Th17细胞分化

检测真正的IgE的表达小鼠B系细胞

鼠标朴素CD4

小鼠树突状细胞亚群的分离的高效和高产的方法

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

IL-22表达淋巴细胞使用记者小鼠的可视化

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

可行的小鼠IL - 17分泌性T细胞的检测和隔离

副本

摘要

探索更多视频

此视频中的章节

隔离和幼稚的CD4 T细胞的Th17细胞分化

检测真正的IgE的表达小鼠B​​系细胞

鼠标朴素CD4

小鼠树突状细胞亚群的分离的高效和高产的方法

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

IL-22表达淋巴细胞使用记者小鼠的可视化

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

检测真正的IgE的表达小鼠B系细胞

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 ⁺ T 细胞的早期分化