Name: quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood
Uploaded: 2012-04-16T13:00:00
Description: 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

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?...

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	<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>

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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

Use of a Monocyte Monolayer Assay to Evaluate FcÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â³ Receptor-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

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

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

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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

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;

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±

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 ...