这项工作由美国国立卫生研究院的支持下批准R15 CA137499 - 01（FB）和来自俄亥俄州立大学（FB）的启动资金的一部分。

1。小鼠股骨和胫骨和骨髓细胞的文化剖析<ol><li>牺牲的CO 2窒息2鼠标和仔细剖析未经切割的骨端的胫骨和股骨。 </li><li>从所有连接的组织，用纸巾清洁的骨头。一定要小心，不要打破骨头。 </li><li>在70％乙醇10分钟在35毫米培养皿中浸泡消毒的骨头。从这一刻起，一个生物安全罩内工作，以避免污染的细胞培养。 </li><li>从乙醇中恢复的骨头，让他们在培养皿内的生物安全柜5分钟空气干燥。 </li><li>削减一半的股骨和胫骨最薄的提示。骨内注入1毫升的RPMI培养基，无菌培养皿上使用无菌注射器（无血清，但用抗生素）。 </li><li>收集细胞悬液，洗涤2X的RPMI培养基在1100 RPM在冷冻离心机（4℃）离心10 min与水平转头15 mL离心管中。 </li><li>最后一次洗涤后，重悬细胞ACK裂解液在2毫升，在室温下5分钟孵育以消除红细胞。 </li><li>加入含10％胎牛血清的RPMI 13毫升，重悬，如1.6中所述的相同的设置，在这个中型的2X清洗。 </li><li>计数细胞，调整至2 × 10 5细胞/ ml的RPMI 10％FBS，并添加RM GM - CSF（20 ng / ml的终浓度）5。 </li><li>添加10毫升这种悬挂无菌，微生物质量，10厘米的培养皿，和文化中的CO 2培养箱（37℃，5％CO 2）。 </li><li>三天后，加入10毫升20毫微克/升rmGM - CSF的RPMI 10％胎牛血清的另一个准备板块。 </li><li>三天后的10 ml的细胞悬液从每个培养皿中恢复，1.6离心，重悬在相同体积的RPMI 10％FBS的rmGM - CSF和返回的培养皿中。细胞培养中的CO 2培养箱为2天。 </li></ol> 2。区议会的收集和标签<ol><li>经过8天的文化松散贴壁细胞恢复洗新鲜培养基的培养皿。此协议呈现约2-4 X10 8区议会在我们手中。流式细胞仪检测细胞可以为DC表型分析。 </li><li>收集细胞，然后655 Qtracker细胞标记试剂盒，严格按照制造商的指示标记。 </li><li>我们得到了优异的成绩，在浓度为10 nm Qdots标签小鼠骨髓来源的区议会。而要做到这一点，Qtracker细胞标记试剂盒组成等分A和B是在等量混合，在室温下5分钟的孵育，并立即稀释震荡30秒1 / 100新鲜培养基</li><li>染色5 × 10 6区议会，混合5μL每个套件的组件A和乙在Eppendorf管中，在室温5分钟孵化。然后，加入1 ml的RPMI 10％FBS的孵化，和30秒的旋涡。 </li><li>加入0.5毫升细胞悬浮液含5 × 10 6区议会，37 ° 60分钟的彗星。 </li><li>然后，洗细胞在RPMI 10％FBS（1100 RPM）的2倍。可以为进一步的文化，或在PBS重悬细胞注射在实验小鼠的RPMI。 </li></ol> 3。标记直流评价<ol><li>通过荧光显微镜观察细胞标记，可以评估。而要做到这一点，一滴细胞悬液加入到载玻片，玻璃盖玻片覆盖，并用荧光显微镜考虑到这些Qdots排放和565nm的405 - 525nm的激发光谱分别评估（ 图3 ） </li><li>在这一点上，可以使用标记的细胞注入动物，或成熟，可诱导这些细胞孵育48小时（37 ° C，5 CO 2）中存在的LPS（100毫微克/毫升）和TNF -α（ 20毫微克/毫升）。荧光标记不会影响DC的成熟（ 图4）。 </li></ol> 4。代表性的成果： 特此，我们介绍了如何准备小鼠骨髓来源的区议会，并标有荧光Qdots图。 1总结了程序，以获取细胞，而图。 2描述了程序标签Qdots他们。 如图所示。 3，几乎所有的细胞被标记此过程中，这是不是DC的成熟与炎症因子（ 图4）图的影响 。 5显示Qdot染色DCS（图5A）的行为类似非染色细胞，与炎症的鸡尾酒治疗。这两种细胞的准备工作同样上调共刺激分子的表达（ 图 5b）;并出示类似IL - 6（ 图 5c）和一氧化氮（图5D）的成熟刺激。这同意与先前类公布的数据表明Qdot染色不会影响DC的能力，变成成熟的细胞，从而引发免疫反应[6]。然后，这些细胞可以用于注射实验动物。 如图所示。 6，术后第2天静脉注射到小鼠体内标记的DC，我们能够发现在脾Qdot染色细胞。这也是与以前公布的数据显示Qdot纳米晶体的使用，以评估的DC在体内的迁移协议[6]通过非侵入性的成像和流式细胞仪。 <img src="/files/ftp_upload/2785/2785fig1.jpg" alt="图1"/> 图1 DC从骨髓中生成的流程图。现将我们描绘的过程中获得的骨髓来源的DC。两个胫骨和股骨的解剖和周围组织的清洁。骨提示保存和内部的骨骼中等刷新。消除红细胞后，骨髓细胞培养8天在GM - CSF的存在，分化成区议会。 <img src="/files/ftp_upload/2785/2785fig2.jpg" alt="图2"/> 图2 标签程序流程图 。 Qtracker细胞标记试剂盒组成的平等等分A和B是一个Eppendorf管中混合。树突状细胞是从8天骨骨髓文化与GM - CSF和收集与标签的染料混合在37C 60分钟。然后细胞被冲在媒体，消除多余的标签颗粒。 <img src="/files/ftp_upload/2785/2785fig3.jpg" alt="图3"/> 图3。 议会荧光显微后立即标签 。标记的细胞下降沉积在载玻片和盖玻片覆盖。然后，样品进行了评估在荧光显微镜和图像通过一个Micropublisher 5.0数字CCD彩色摄像机（Qimaging，加拿大不列颠哥伦比亚省萨里）收购。 <img src="/files/ftp_upload/2785/2785fig4.jpg" alt="图4"/> 图4 48 h后，在体外成熟的区议会的荧光显微。培养48小时与LPS（100毫微克/毫升）和肿瘤坏死因子-α（20毫微克/毫升），以CO 2培养箱（37℃，5％的CO 2）的玻片上的标记区议会。然后，样品分别洗净，用PBS（5分钟，2X）（15分钟，4 ° C），用丙酮固定，并用DAPI counterstained。细胞在荧光显微镜上述评价。 <img src="/files/ftp_upload/2785/2785fig5.jpg" alt="图5"/> 图5。Qdot 染成熟DC的生物活性。上述体外成熟的标记区议会进行了分析流式细胞仪（一）及（B） 。 （一）Qdot染的细胞会在FL3（PercP）通道的一个强烈的信号。直方图：不染控制阴影。 （二）Qdot染色和未染色区议会此外对成熟的标志物CD86和CD40的特异性抗体和同型对照染色。细胞，然后分析在FACSort流式细胞仪（Becton Dickinson公司，加利福尼亚州圣何塞）。此外，IL - 6（C）和一氧化氮（NO）测定上清成熟Qdot染区议会和控制。通过ELISA分析和Griess检测正如我们前面描述的7，8，IL - 6和一氧化氮水平测定。所有在此图中显示的数据是代表两个或三个独立的实验显示了类似的结果。数据是通过方差分析，使用的GraphPad软件。 <img src="/files/ftp_upload/2785/2785fig6.jpg" alt="图6"/> 图6。 检测标记在解剖组织的区议会。成熟DCs标记（1 × 10 6细胞）静脉注射到C57BL / 6小鼠。 （一）两天后，小鼠处死，脾脏收集，管理单元冻结，OCT包埋，8微米的部分使用低温恒温器。然后，样品固定，用丙酮（15分钟，4 ° C），用DAPI counterstained。细胞在荧光显微镜上述评估图6A：40倍的放大倍率， 图6B：100X的放大倍率， 图。 6C，400X的放大倍率。

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小鼠髓系）区议会已被广泛应用于以DC为基础的疫苗，以确定疗效和改善;调查DC：T细胞相互作用或DC发展; 在 9-11各种疾病，并确定它们的作用。现将我们展示了如何从从胫骨和股骨的骨髓恢复的前兆生成区议会。我们的骨头，没有切割提示恢复，使我们能够消毒浸没在乙醇70％，从而减少污染的可能性。为了从骨髓细胞分化议会，我们只用了 5先前所描述的GM - CSF的。尽管有些协议还使用IL - 4，据报道，这种因子是与GM - CSF的水平高12的工作时，没有必要。事实上，我们先前已经表明，这些区议会能诱导免疫反应 13 。此外，应采取恢复为期8天的文化的洗涤培养基的培养皿中，只是笼统地贴壁细胞，因为贴壁细胞表现出更多的类似单核细胞表型。在这里，我们展示区议会与荧光Qdot颗粒标签。这种标签有一些优势，尊重其他方法。首先，Qdots颗粒细胞很容易被纳入。二，荧光信号是非常高的，不是由DC的成熟而改变。三，荧光的细胞或组织固定溶剂，如丙酮，相反会发生什么，如果GFP是用来标记 14区议会，给予更多的时刻选择染色协议的灵活性时不会丢失。最后，尽管这些粒子的高荧光信号允许组织自体荧光的细胞可视化。如前所述6，Qdot染色并不影响这些细胞的成熟能力。现将我们Qdot染色DCS的行为在非染DCS类似的方式，上调共刺激分子，IL - 6和一氧化氮在炎症刺激生产。虽然区议会专门触发免疫反应的细胞，它们已被证明参与在病理条件下，如癌症和动脉粥样硬化4，15，16 。他们还声称，参与血管生成过程中甚至建议为参与结构，在发展的新船19，20 17，18 ，。因此，允许直流跟踪体内，并确定他们在不同的组织4，21的地域本地化的方法 ，22个是非常有价值的。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> C57BL / 6小鼠</td><td>杰克逊实验室</td><td></td><td>雌性，6〜8周龄</td></tr><tr><td>的RPMI </td><td> Invitrogen公司</td><td> 11875-119 </td><td></td></tr><tr><td>胎牛血清，合格</td><td> Invitrogen公司</td><td> 10437-028 </td><td></td></tr><tr><td>抗生素的antimycotic </td><td> Invitrogen公司</td><td> 15240-096 </td><td></td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 10010-049 </td><td></td></tr><tr><td> ACK裂解缓冲</td><td>龙沙Walkersville，公司</td><td> 10 - 548E </td><td></td></tr><tr><td>重组小鼠GM - CSF的</td><td> PeproTech公司</td><td> 315-03 </td><td></td></tr><tr><td> Qtracker 655细胞标记试剂盒</td><td> Invitrogen公司</td><td> Q25021MP </td><td></td></tr><tr><td>脂多糖</td><td> Invivogen </td><td> tlrl - eblps </td><td></td></tr><tr><td>重组小鼠肿瘤坏死因子α </td><td> Peprotech </td><td> 315 - 01A </td><td></td></tr><tr><td> CD86抗体</td><td> BD公司</td><td> 553691 </td><td></td></tr><tr><td> CD40抗体</td><td> BD公司</td><td> 553791 </td><td></td></tr><tr><td> Griess试剂系统</td><td> Promega公司</td><td> G2930 </td><td></td></tr><tr><td> IL - 6的捕获抗体</td><td> eBioscience公司</td><td> 13-7061-81 </td><td></td></tr><tr><td> IL - 6的检测抗体</td><td> eBioscience公司</td><td> 13-7062-81 </td><td></td></tr></tbody></table>

generation and labeling of murine bone marrow-derived dendritic cells with qdot nanocrystals for tracking studies

树突状细胞（DC）的专业抗原提呈细胞（APCs）在外围组织和免疫器官，如胸腺，骨髓，脾，淋巴结和Peyer氏斑 1-3发现。区议会目前在外围组织样本的有机体抗原的存在，他们采取了，过程，并在其表面主要组织相容性分子（MHC）的背景下出现在。然后，抗原加载DCS迁移到他们目前处理的抗原引发特异性免疫反应的T淋巴细胞的免疫器官。评估区议会的迁徙能力的方法之一是标签与荧光染料 4 。 我们特此证明Qdot荧光纳米晶体的使用标签小鼠骨髓来源的DC。这种标签的优点是，Qdot纳米晶体具有稳定和持久的荧光检测标记细胞，恢复组织，使他们的理想。而要做到这一点，第一个细胞将恢复从小鼠骨髓培养8天粒细胞巨噬细胞集落刺激因子的存在，以诱导DC分化。这些细胞就会然后用荧光Qdots标记，在体外孵化。用荧光显微镜，可以可视化染色细胞。细胞可注入实验动物，在这一点上，或可以成为成熟的细胞在体外孵化后炎症刺激。在我们手中，DC的成熟并没有确定的荧光信号的损失也不Qdot染色影响区议会的生物学特性。注射后，这些细胞可以通过荧光显微镜下面的典型解剖和固定程序，免疫器官。

Watch this Scientific Journal Video about Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies at JoVE.com

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

树突状细胞摄取抗原和迁移对目前处理的T细胞抗原的免疫器官。 Qdot纳米晶体的标签提供了一个长期和稳定的的荧光信号。这使得跟踪树突状细​​胞的荧光显微镜的不同器官。

小鼠骨髓来源的树突状细胞与跟踪研究的Qdot纳米晶的生成及标签

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

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Research

JoVE Journal

Immunology and Infection

24.7K 次观看.  Ohio University. 树突状细胞摄取抗原和迁移对目前处理的T细胞抗原的免疫器官。 Qdot纳米晶体的标签提供了一个长期和稳定的的荧光信号。这使得跟踪树突状细​​胞的荧光显微镜的不同器官。

视频: Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

使用骨髓源性巨噬细胞巨噬细胞极化的调查

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

科研

免疫与感染

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

骨髓衍生的巨噬细胞产

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

隔离和小鼠骨髓的静脉注射单核细胞衍生

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

不成熟，成熟和耐受性树突状细胞的生成与代谢不同表型

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

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 ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

利用新的细胞分选策略, 从小鼠骨髓中生成大量的髓样祖细胞和树突状体细胞前体

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

小鼠骨髓源性巨噬细胞 Inflammasome 活化和 Pyroptotic 细胞死亡的检测

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...