这项工作是支持的一部分，由美国国立卫生研究院（N01，HHSN272201100019C，19岁以下人工智能089992，NCRR / GCRC计划M01-RR00125的）。作者声明没有竞争的金融利益，并感谢的马克Shlomchik博士，耶鲁大学的细胞分选仪的核心设施。

1。免疫细胞的分离与激励<ol><li>获得书面知情同意的当地机构审查委员会的指引下健康志愿者后10-50毫升的肝素化血。必须明确确定每个志愿者或患者的发病机制，排除和纳入标准老化或疾病的研究。 </li><li>分离人外周血单个核细胞（PBMC）使用彩管管或Ficoll分离，Paque另外根据制造商的说明（GE Healthcare公司，新泽西州）和隔离7（试剂， 表1）天进行实验。 </li><li>孵育1.5毫升0.6毫升培养基单独管或刺激的TLR配体或其他活化剂，如单核细胞（3×10 6 /管）TLR7 / 8配R848（0.6毫升中加0.6μL10毫米TLR7 / 8配R848，最终在37°C浓度10μM）为10-60分钟（InvivoGen，加利福尼亚州）9。 </li></ol> 2。实验室鄂陵湖的细胞<ol><li>标签使用荧光标记的抗体在活细胞悬浮细胞系和其他表面标志。标签3×10 6单核细胞为20分钟，4°C在100μl的PBS / 2％FBS的1.5毫升的Eppendorf管表面抗体的单核细胞或DC谱系（ 见表2）具体的鸡尾酒。初步实验和样品应分批处理，以尽量减少很多，很多的变化，可确定最佳抗体稀释。供应商抗体股票0.1毫克/毫升用5微升抗体（1:20稀释）。使用单独的细胞，只有一个单一的颜色结合，设立仪器上的荧光渠道标记的管。 </li><li>经过表面标记，细胞固定在4％PFA / PBS在室温为10分钟。对于存储，直到时间的分析，离心机500 XG和重悬细胞在冻结缓冲区（90％FBS的含10％DMSO）在-80°C间细胞，直到一天的检测。 </li><li>对于评估过程中分批Øf未处理和刺激细胞从一组捐助者一起，以尽量减少变异。在分析当天，解冻细胞很快在37°C水浴，离心机500XĞ清除冻结的缓冲区。 </li><li>通透细胞检测细胞内细胞因子或其他标记，重悬细胞悬液100μL的BD彼尔姆/洗涤缓冲液（BD Biosciences公司，新泽西州）。例如，1:20稀释的兔抗NF-κB的（P65）抗体（终浓度为10微克/毫升，圣克鲁斯生物技术，加州）在室温20分钟，在500 XG离心，吸出上清液和细胞内的信号元件的标签重悬细胞100μL屋宇署权限/洗涤缓冲液1:250稀释的羊抗兔IgG-Alexa647（Invitrogen公司，加利福尼亚州）和20分钟在室温下孵育。 </li><li>立即成像，前染液细胞核用DAPI（0.2微克/毫升，Invitrogen公司，加利福尼亚）或碘化（有价证券; 20毫微克/毫升，Invitrogen公司，加利福尼亚）。 </li></ol> 3。 ImageStream分析<ol><li>批次样品进行成像技术，以减少人类样本之间的变异ImageStream。功率的激光仪器设置如下：200毫瓦为488纳米，10毫瓦为658纳米，405纳米和250兆瓦。 </li><li>分析数据文件（ 图1），第一门事件与正常PI强度和高PI宽高比（宽：高比例），以区分单细胞碎片（低强度）有价证券或多细胞活动（R1）（有价证券投资高强度和低展弦比）。单核细胞内细胞CD14阳性细胞的大门。 MDCS是在一个细胞，CD11c和低CD4（R）和林1标记（4915）也低高门。 </li><li>用意念软件（Amnis，WA）提供一个有效的每个细胞的活化程度的定量测量。确定细胞质转录因子NF-κB核染料PI（或合作本地化）的相似性表明易位进入细胞核（R4）的。一个高NF-κB/PIlocali相关zation体现在一个高的相似性得分，并表示激活10度。 </li><li>比较不同的治疗组患者人群，表明细胞的功能相对效率之间的相似性比率。通过绝对值或倍差异的统计比较量化的样本之间的数据。用意念从人口直方图的关键环节（ 图2）显示细胞图像的软件。 </li></ol> 4。代表结果我们TLR7 / 8配，R848（ 图1）刺激单核细胞的信号转导通路在病毒配体模型量化。我们收集的细胞定位图像和人口统计数字，在我们的样本组（ 图2）。 ImageStream让我们门细胞亚群，直接从单核细胞和图像细胞的反应，从门，但无序的细胞群。在这里，我们量化的TLR小号的影响ignaling核定的NF-κB（P65）在林1，CD4 +暗淡，素CD11c +髓区议会（MDCS）。基线时，我们发现高比例的未刺激细胞转录因子在细胞质中的NF-κB（P65）。刺激后，细胞治疗有显着较高的相似性的NF-κB核染色的PI得分（P65），刺激后的NF-κB（P65）易位进入细胞核（中位数的相似性得分分别为1.52和3.01， 图。 2）。类似的结果指出在TLR5的刺激单核细胞9。 <img alt="图1" src="/files/ftp_upload/3741/3741fig1.jpg" /> 图1。染色和的浇注战略量化对人类MDCS的刺激作用 。转录因子NF-κB的调节许多免疫系统的基因表达。本研究措施，在从一个有代表性的主题MDCS TLR7 / 8西甲后核定的NF-κB（P65）<html>第二刺激。在集中单一细胞进行鉴定，门上高核长宽比（R1）的PI阳性事件。 MDCS（林1，CD4dim，素CD11c +）在R3门。核本地化的NF-κB（P65）在R4绘制。 <a href="http://www.jove.com/files/ftp_upload/3741/3741fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/3741/3741fig2.jpg" /> 图2。 MDCS刺激后NF-κB的易位，易位进入细胞核（R4）的刺激后的NF-κB（P65）被描绘在未处理和刺激MDCS种群，在未经处理的样品的相似性得分中位数是1.52和3.01刺激的样品（一）。未经处理或R848刺激的细胞群，同时收集的数字图像显示易位进入细胞核（二）代表细胞和NF-B的强度。_upload/3741/3741fig2large.jpg“目标=”_blank“&gt;点击这里查看大图。

<ol>
	<li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5, 133-139 (2004).</li><li>Shaw, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+Human+Toll-like+Receptor+Function+in+Aging.">Dysregulation of Human Toll-like Receptor Function in Aging.</a> Ageing Research Reviews. 10, 346-353 (2011).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nature Rev. Immunol. 1, 135-145 (2001).</li><li>van Duin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+Defect+in+Human+TLR1+Function+and+Expression.">Age-associated Defect in Human TLR1 Function and Expression.</a> J. Immunol. 178, 970-975 (2007).</li><li>Panda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+decrease+in+TLR+function+in+primary+human+dendritic+cells+predicts+influenza+vaccine+response.">Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.</a> J. Immunol. 184, 2518-2527 (2010).</li><li>Kong, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+TLR3+impairs+the+innate+immune+response+to+West+Nile+virus+in+the+elderly.">Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.</a> J. Virol. 82, 7613-7623 (2008).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+interferon+signaling+in+dendritic+cells+from+older+donors+infected+in+vitro+with+West+Nile+virus.">Impaired interferon signaling in dendritic cells from older donors infected in vitro with West Nile virus.</a> J. Infect. Dis. 203, 1415-1424 (2011).</li><li>Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+in+immunobiology.">NF-kappaB in immunobiology.</a> Cell research. 21, 223-244 (2011).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+elevation+in+TLR5+leads+to+increased+inflammatory+responses+in+the+elderly.">Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.</a> Aging Cell. 11, 104-110 (2011).</li><li>George, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+nuclear+translocation+events+using+similarity+analysis+of+multispectral+cellular+images+obtained+in+flow.">Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.</a> Journal of immunological. 311, 117-129 (2006).</li></ol>

没有利益冲突的声明。

ImageStream用于翻译研究的关键步骤是选择相关的对照组和特异性抗体的优化和验证。主题标记的目标群体之间的差异是显而易见的，通过直方图和细胞图像。结合深入分析了有关的受体和信号通路的变化，这些数据将提供宝贵的见解机制的基础免疫失调，在专门的同伙。该技术将受到足够的亲和力有关目标的特异性抗体的可用性。此外，该仪器是最适合多用户设施和分析软件，需要掌握一些护理?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Ficoll分离，加Paque </td><td> GE医疗集团</td><td> 17-1440-03 </td></tr><tr><td> R848 </td><td> invivogen </td><td> tlrl-r848 </td></tr><tr><td>多聚甲醛（16％） </td><td>电子显微镜科学</td><td> 15710 </td></tr><tr><td>屋宇署权限/洗</td><td> BD公司</td><td> 554723 </td></tr><tr><td>细胞CD14-PE </td><td> eBioscience </td><td> 12-0149-41 </td></tr><tr><td> CD4 - PacBlue </td><td>屋宇署Pharmingen公司</td><td> 68436 </td></tr><tr><td>宗族鸡尾酒1（林1）-FITC标记</td><td> BD公司</td><td> 340546 </td></tr><tr><td>素CD11c-PE </td><td> BD公司</td><td> 347637 </td></tr><tr><td> CD123-PE </td><td> BD公司</td><td> 340545 </td></tr><tr><td>兔抗NF-κB（P65） </td><td>圣克鲁斯</td><td> SC-372 </td></tr><tr><td> alexa647 F（AB&#39;）2羊抗兔IgG </td><td> Invitrogen公司</td><td> A21246 </td></tr><tr><td>的DAPI </td><td> Invitrogen公司</td><td> D21490 </td></tr><tr><td> ImageStream X </td><td> amnis </td><td></td></tr></tbody></table> 表1。特定的试剂和设备。 <table border="1"><tbody><tr><td> 细胞类型 </td><td> FITC标记 </td><td> 体育 </td><td> 网站647 </td><td> PacBlue </td></tr><tr><td>单核细胞</td><td></td><td> CD14的</td><td>核因子-κB（P65） </td><td></td></tr><tr><td>的mDC </td><td>林1 </td><td> CD11c的</td><td>核因子-κB（P65） </td><td>的CD4 </td></tr><tr><td> pDC的</td><td>林1</td><td> CD123 </td><td>核因子-κB（P65） </td><td>的CD4 </td></tr></tbody></table> 表2。抗体的细胞信号通路的血统染色面板单核细胞：细胞CD14 + MDC：林1，CD4dim，素CD11c + PDC：林1，CD4dim，CD123 + 林1（系）的标记包括CD3 +，CD14，CD19 +，CD20分子，CD56 +单核细胞，NK细胞，T，B细胞标记。

quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood

在免疫状况的个体差异决定的反应，感染和疾病的严重程度和结果作出贡献。老龄化与病毒和细菌感染的易感性增加和减少记录在体液下降以及细胞介导的免疫反应1,2疫苗的反应。我们最近评估Toll样受体（TLRs），检测微生物感染和触发抗菌宿主防御反应3的先天免疫系统的关键部件老化的影响。在一个健康的器官捐献者的大型队列中，我们发现，从老人的外周血单核细胞减少某些Toll样受体4和减少类似的TLR水平和树突状细胞信号反应（DCS）的表达和功能，抗原提呈细胞，关键在5先天免疫和适应性免疫之间的联系。我们已经表明巨噬细胞中TLR3的失调低层D老人捐助者的区议会感染西尼罗河病毒6,7干扰素生产。 我们的免疫衰老的理解和干预治疗的最重要的是一种特定的细胞响应类型的详细了解和信号转导机制（S）。主要通过流式细胞仪或免疫细胞和测量细胞标记成像，通过传统的免疫反应的研究进展显着，我们的理解然而，这些研究一般限于小样​​本量可从患者和无法进行多个复杂的实验室技术，技术上人体标本。 ImageStream同时高分辨率数字成像结合定量流式细胞仪，从而促进病人易感性的高效捕获contemporaneously调查在多个细胞群。在这里，我们展示的ImageStream在区议会的使用，以评估TLR7 / 8磷酸化和核易位的一个关键转录因子NF-κB，从而启动众多8免疫反应的关键基因的转录激活介导的增加。使用这种技术，我们最近也证明先前未确认变更的TLR5的信令和捐助者从旧的单核细胞NF-κB信号通路，可能有助于改变老化9免疫反应。

Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

我们描述ImageStream技术的使用（<a href="http://www.amnis.com/" target="_blank"&gt; www.amnis.com</a&gt;），它结合了定量流式细胞仪同时高分辨率数字成像，量化定义良好的病人同伙的主要免疫细胞的细胞机制。我们的研究提供了一个蓝图转化调查量化谱系在小样本的具体细胞的反应，受同伙。

定量成像小样本人体血液中单核细胞和树突状细胞系特定的Toll样受体介导的信号

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

quantitative-imaging-lineage-specific-toll-like-receptor-mediated

Research

JoVE Journal

Immunology and Infection

35.0K 次观看.  Yale University School of Medicine. 我们描述ImageStream技术的使用（ www.amnis.com），它结合了定量流式细胞仪同时高分辨率数字成像，量化定义良好的病人同伙的主要免疫细胞的细胞机制。我们的研究提供了一个蓝图转化调查量化谱系在小样本的具体细胞的反应，受同伙。

视频: Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

在调节Toll样受体激活Neu1唾液酸酶活动的检测

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

科研

免疫与感染

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

小鼠腹腔巨噬细胞的分离，以贯彻基因表达分析后Toll样受体刺激

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

单核细胞单层试验中用于评估Fcγ受体介导的吞噬作用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

为了应对抑菌活性的实时成像人力初级中性粒细胞和单核细胞<em&gt;一种。曲霉</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

人干扰素α超灵敏单分子阵列数字酶联免疫吸附试验的研制与验证

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...