我们非常感谢他们的宝贵贡献，K. Nakagaki，博士阁下石井，铃木博士光，A.山形，光Oofusa。

1。生产和净化的sGMRα <ol><li>从人胎盘cDNA文库中扩增cDNA的聚合酶链反应（PCR）的sGMRα。 </li><li> 50基地5&#39; - UTR序列杆状病毒多角月底和27基编码的RGS His - tag的3&#39;末端的另一个PCR扩增PCR扩增。 </li><li>插入质粒pMSG1.1MG的扩增的PCR产物。 </li><li>注入鸡蛋蚕PND - W1株pHA3PIG助手载体的建设psGMR/M1.1MG。 </li><li>后部孵化G0期幼虫蛾25 ° C。 G1的屏幕获得通过交配，兄弟姐妹之间或与PND - W1在眼里MGFP表达，获得转基因蚕轴承sGMRα基因的胚胎。 </li><li>从转基因蚕的蚕茧中提取重组sGMRα与含有500MM氯化钠的磷酸盐缓冲和搅拌在4 ° 24小时的彗星。 </li><li> 20000克为15分钟离心样品删除沉淀。 </li><li>应用上清镍亲和层析柱。 </li><li>洗净咪唑10MM，500MM氯化钠和20mm磷酸钠，pH6.3，与咪唑从10mm到250mm的线性渐变洗脱列。池的分数包含对纯化sGMRα和透析磷酸盐缓冲液（PH7.4）（PBS）的。 </li></ol> 2。生物素的重组GM - CSF的<ol><li>删除对PBS透析山梨醇准备。 </li><li>加入1ml至20mm的冷钠元碘酸溶液，在4 ° C的冰的解决方案。 </li><li>孵育30分钟就在黑暗中的冰样本。 </li><li>添加甘油的终浓度为15mm的解决方案。 </li><li>透析对100MM醋酸钠缓冲液（PH5.5）的解决方案</li><li>生物素酰肼加入终浓度为5mm的解决方案，并不断搅动解决在室温2H。 </li><li>取出透析对PBS，PH7.4未反应的物质。 </li></ol> 3。不含细胞的GM - CSF受体配体结合法<ol><li>大衣与单克隆抗多聚组氨酸抗体50μL一夜之间4微孔板° C。 </li><li>用PBST500μL洗5次。 </li><li>添加阻塞的解决方案和孵化为3h 4 ° C。100μL </li><li>用PBST500μL洗5次。 </li><li>在封闭液中添加sGMRα50μL，4℃过夜孵育° C。 </li><li>用PBST500μL洗5次。 </li><li>样本含有GM - CSF的自身抗体和生物素化的GM - CSF的25μl加入25μl。孵育1h后在4 ° C至形成sGMRαGM - CSF复杂。 </li><li>用PBST500μL洗5次。 </li><li>添加0.4mm的链霉亲和素碱性磷酸酶1小时50μL4 ° C至检测生物素化的GM - CSF的约束sGMRα。 </li><li>用PBST500μL洗5次。 </li><li>解决方案，并在室温下孵育1h后加入化学发光底物50μL。 </li><li>使用化学发光酶标仪，检测化学发光活动。 </li></ol> 4。代表性的成果: 在GM - CSF受体信号转导通路的第一步是GM - CSF的结合，GM - CSF的受体细胞表面上的。因为GM - CSF的抗体特异性结合GM - CSF和块及其受体在体外16,17的约束力，我们推测，自身抗体抑制这种直接绑定到GM - CSF的第一反应。如前所述（编号9-15），我们应用转基因家蚕的技术来获得大量高纯度的重组sGMRα。 加载在SDS - PAGE非还原条件​​下，当蚕衍生的重组sGMRαsGMRα显示，两个单体（45 kDa）的和二聚体（90 kDa）的形式，而只有单体的形式还原条件下检测到（图1B） ，表明重组sGMRα混合单体和二硫键连接调光器18。 使用无细胞系统（图2A），我们评估sGMRα抑制GM - CSF的结合，GM - CSF的抗体（图2B和图2C）。浦野等，这些方法在参考文献19描述。结合抑制，不同浓度的GM - CSF的抗体（图3A）19（R = 0.988，P = 0.002）密切相关的生长抑制。同样，结合抑制与生长的抑制作用显着相关。结合抑制GM - CSF的自身抗体（图3B）19在剂量依赖性增加。这些来自不同患者的血清IgG组分两个参数相互呈正相关（R = 0.589 p = 0.006，图3c）。无论是结合抑制，也不生长抑制与Kd值，因此，这两个参数是通过具有约束力的亲和力 19日的影响。因此可重复性的约束力和G之间的数据通过三个独立的实验对三种不同的样品获得的rowth抑制了评估。的变异系数表示，更优良的生物活性（表1）无细胞系统。 <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="图1" /> 图1。sGMRα使用转基因蚕生产过程流程图。一）结构转化载体9-11。二）SDS - PAGE和考马斯亮蓝染色的减少和非还原条件​​下纯化sGMRα。 sGMRα，可溶性GM - CSF受体α; MGFP，怪物绿色荧光蛋白;，5&#39; -非翻译区序列家蚕核型多角体病毒多角体BmNPVpol5&#39; UTR区; SV40的多聚腺苷酸，SV40的多聚腺苷酸信号序列，P 3xP3，3xP3推动者， P ser1，ser1子; HR3， 有机农业研究所 ，素L -链多聚腺苷酸信号序列多聚腺苷酸; 家蚕核型多角体病毒HR3增强。 <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="图2" /> 图2无细胞检测系统的计划。 A）竞争结合实验，使用由蚕生产sGMRα的。二）中和非中和抗体的影响，对无细胞系统的结合抑制。三）区别不同浓度的中和抗体和非中和抗体之间的结合抑制。 GM - CSF，粒细胞 - 巨噬细胞集落刺激因子;sGMRα，可溶性GM - CSF受体α;他，RGS - His - tag的美联社，碱性磷酸酶。 <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="图3" /> 图3，GM - CSF的结合抑制sGMRα由GM - CSF的多克隆抗体或自身免疫性PAP患者的血清IgG分数的影响。一）和GM - CSF的抗体结合抑制生长的抑制之间的关系。二）在不同浓度的GM - CSF抗体的结合抑制。三）集成电路50％的结合抑制和血清IgG 分数 19％的增长抑制之间的关系。 IC，50％抑制浓度50; GM - CSF，粒细胞-巨噬细胞集落刺激因子。 <table border="1"><tbody><tr><td>样品</td><td>一</td><td>乙</td><td>彗星</td></tr><tr><td colspan="4">批间</td></tr><tr><td> ＃的测定</td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td>平均值（％结合抑制） </td><td> 61.6 </td><td> 63.8 </td><td> 69.7 </td></tr><tr><td>平均值（％生长抑制） </td><td> 21.0 </td><td> 73.4 </td><td> 82.8 </td></tr><tr><td>变异系数（％）（％约束力抑制） </td><td> 6.0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td>变异系数（％）（％的增长，抑制） </td><td> 64.4 </td><td> 18.3 </td><td> 10.4 </td></tr></tbody></table>两种检测均在5纳克/毫升的GM - CSF和GM - CSF的抗体的浓度等于。 表1％的约束性和增长禁忌之间的变化系数通过3次独立实验的比较。

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无细胞检测估计具有优良的重现性和快速性的GM - CSF的自身抗体的中和能力。结合抑制GM - CSF的自身抗体或患者的血清IgG分数是评估这个实验。数据显示，结合抑制细胞免费检测和使用TF - 1细胞，分别为生物活性的生长抑制之间的关系。已被广泛使用的生物活性，但窝藏在比较数据之间不同的设施和不同的时间点，我们通过使用这个新的系统可避免的困难。 GM - CSF的结合sGMRα?...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

背景:在此之前，我们表明，中和能力，但没有GM - CSF的抗体浓度是在患者自身免疫性肺泡蛋白沉积症（PAP）的1-3疾病的严重程度相关。废止的GM - CSF在肺癌的生物活性可能导致自身免疫性PAP 的 4,5，它是有前途的GM - CSF自身抗体的中和能力的评估与人民行动党在每个病人的病情轻重程度来衡量。 到现在为止，已评定的GM - CSF自身抗体的中和能力评估人类骨髓细胞或TF - 1 细胞 6-8与GM - CSF的刺激的生长抑制。然而，在生物测定系统，它往往是问题，以获得可靠的数据，以及比较来自不同实验室的数据，由于技术上的困难，保持在一个恒定的条件下的细胞。 目的:为了模仿GM - CSF，GM - CSF受体细胞表面上的具有约束力的使用无细胞受体结合测定。 方法:转基因家蚕技术是获得高纯度9-13的重组可溶性GM - CSF受体α（sGMRα）为大量的应用。重组sGMRα载而不融合丝蛋白在亲水丝胶丝线层，因此，我们可以很容易地从良好的纯度蚕茧14,15与中性水溶液中提取。幸运的是，寡糖的结构，这是与GM - CSF结合的关键，更类似于人类sGMRα的结构比其他昆虫或酵母生产。 结果:无细胞检测系统使用sGMRα取得的数据，具有很高的可塑性和可靠性。 GM - CSF的剂量依赖性绑定sGMRα以类似的方式使用TF - 1细胞的生物活性，抑制多克隆GM - CSF的抗体，这表明我们的新的无细胞检测系统使用sGMRα是中和活性的测量更为有用GM - CSF的自身抗体比使用TF - 1细胞或骨髓细胞的生物测定系统。 结论:我们建立了无细胞的检测，量化的GM - CSF抗体的中和能力。

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

我们设计了一个无细胞受体结合实验，以估计约束力的粒细胞 - 巨噬细胞集落刺激因子（GM - CSF的）受体。它使我们能够评估竞争性抑制GM - CSF的抗体生物素标记的GM - CSF的可溶性GM - CSF受体α的结合，具有优良的重现性。

一个细胞检测系统估算的GM - CSF抗体的中和能力，采用重组可溶性GM - CSF受体

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

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Immunology and Infection

12.6K 次观看.  Niigata University Medical and Dental Hospital. 我们设计了一个无细胞受体结合实验，以估计约束力的粒细胞 - 巨噬细胞集落刺激因子（GM - CSF的）受体。它使我们能够评估竞争性抑制GM - CSF的抗体生物素标记的GM - CSF的可溶性GM - CSF受体α的结合，具有优良的重现性。

视频: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

生物能量和氧化突发:协议用于人类白细胞和血小板的隔离与评价

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

科研

免疫与感染

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (tHgm) 细胞

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in na&#239;ve human populations.

This protocol details an investigation of the early interactions between virally infected nasal epithelial cells and innate cell activation. Individual subsets of immune cells can be distinguished based on their activation in response to viral infections. They can then be further investigated to determine their effects on early antiviral responses.

用于研究呼吸道病毒感染期间先天免疫细胞活化的非接触式共培养模型

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The ...

Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The increasing number of SARS-CoV-2 infections raises the need for better antibody detection assays. Current antibody detection methods compromise sensitivity for speed or are sensitive but time-consuming. A large proportion of SARS-CoV-2-neutralizing antibodies target the receptor-binding domain (RBD), one of the primary immunogenic compartments of SARS-CoV-2. We have recently designed and developed a highly sensitive, bioluminescent-tagged RBD (NanoLuc HiBiT-RBD) to detect SARS-CoV-2 antibodies. The following text describes the procedure to produce the HiBiT-RBD complex and a fast assay to evaluate the presence of RBD-targeting antibodies using this tool. Due to the durability of the HiBiT-RBD protein product over a wide range of temperatures and the shorter experimental procedure that can be completed within 1 h, the protocol can be considered as a more efficient alternative to detect SARS-CoV-2 antibodies in patient serum samples.

The outlined protocol describes the procedure for producing the HiBiT-receptor-binding domain protein complex and its application for fast and sensitive detection of SARS-CoV-2 antibodies.

使用基于HiBiT的生物报告器检测SARS-CoV-2受体结合域抗体

The emergence of the COVID-19 pandemic has increased the need for better serological detection methods to determine the epidemiologic impact of severe ...