| Cytokine-Stimulated Phosphoflow of Whole Blood Using CyTOF Mass Cytometry
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Author:
Date:
2015-06-05
[Abstract] The ability to assess the function of a range of cytokine, antigen receptor, and Toll-like receptor (TLR) signaling pathways in a range of immune cells could provide a kind of fingerprint of the state of the human immune system. The mass cytometry or CyTOF, platform allows for the parallel application of about 40 labeled antibodies to a single sample, creating the possibility to read out many cell types and signaling pathways in a single small blood sample. We developed such a mass cytometry panel, consisting of 22 antibodies to cell surface lineage markers and 8 antibodies to phospho-specific epitopes of signaling proteins. These antibodies were chosen to discriminate all major white blood cell lineages, to a level of detail that includes subsets such as naïve, central memory, effector ...
[摘要] 在一系列免疫细胞中评估一系列细胞因子，抗原受体和Toll样受体（TLR）信号通路的功能的能力可以提供人免疫系统状态的一种指纹。质谱仪或CyTOF平台允许将约40种标记的抗体平行应用于单个样品，产生在单个小血液样品中读出许多细胞类型和信号传导途径的可能性。我们开发了这样的大规模细胞仪板，由22个抗体的细胞表面沿袭标记和8抗体的磷酸特异性表位的信号蛋白。选择这些抗体以区分所有主要白细胞谱系，其细节水平包括诸如初始，中枢记忆，效应记忆和晚期效应CD4 +和CD8 + T细胞，天然，过渡和转换记忆B细胞的子集，浆母细胞，骨髓和浆细胞样树突状细胞，CD16 +和CD16 + CD56 + NK细胞，CD16 +和经典单核细胞等。在我们的标准门控方案中定义这样的细胞亚群。选择8种磷酸特异性抗体以代表响应细胞因子，TLR和抗原受体信号传导的主要信号转导节点。该抗体组与8种标准刺激条件（未刺激，IFNa，IL-6，IL-7，IL-10，IL-21，LPS，PMA +离子霉素）一起使用，尽管可以加入其它刺激。健康对照与具有未知病因的免疫缺陷的受试者的比较可能有助于阐明这种缺陷的机制。
酪氨酸，丝氨酸和苏氨酸残基的磷酸化对于控制参与各种细胞事件的蛋白质活性是至关重要的。激酶和磷酸酶的分类调节许多不同的细胞信号传导途径，例如T和B细胞信号传导，调节细胞凋亡，生长和细胞周期控制，加上那些参与细胞因子，趋化因子和应激反应的细胞内蛋白磷酸化。 ...
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| Cytokine-stimulated Phosphoflow of PBMC Using CyTOF Mass Cytometry
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Author:
Date:
2015-06-05
[Abstract] Phosphorylation of tyrosine, serine, and threonine residues is critical for the control of protein activity involved in various cellular events. An assortment of kinases and phosphatases regulate intracellular protein phosphorylation in many different cell signaling pathways. These pathways include T and B cell signaling, regulating growth and cell cycle control, plus cytokine, chemokine, and stress responses. Phosphoflow assays combine phosphoprotein-specific antibodies with the power of flow cytometry to enhance phosphoprotein study. In our assay, peripheral blood mononuclear cells are stimulated by cytokines, fixed, surface-stained with a cocktail of antibodies labeled with MAXPAR (brand name) metal-chelating polymers and permeabilized with methanol. They are then stained with ...
[摘要] 酪氨酸，丝氨酸和苏氨酸残基的磷酸化对于控制参与各种细胞事件的蛋白质活性是至关重要的。各种激酶和磷酸酶调节许多不同细胞信号传导途径中的细胞内蛋白磷酸化。这些途径包括T和B细胞信号传导，调节生长和细胞周期控制，加上细胞因子，趋化因子和应激反应。 Phosphoflow测定结合磷蛋白特异性抗体与流式细胞术的力量，以增强磷蛋白研究。在我们的测定中，外周血单核细胞被细胞因子刺激，固定，用用MAXPAR（商品名）金属螯合聚合物标记并用甲醇透化的抗体的混合物表面染色。然后用细胞内磷酸特异性抗体染色。
我们使用CyTOF TM 质谱仪获取ICP-MS（电感耦合等离子体质谱）数据。选择的当前质量窗口大约是AW 103-203，其包括用于大多数抗体标记的镧系元素，以及用于DNA嵌入剂的铱和铑。使用FlowJo软件的双计数信号数据的后续分析允许基于每个质量通道中的双计数信号来分析细胞类型。确定每种细胞类型的百分比，并报告为父细胞类型的百分比。报道中值以定量响应刺激的每种蛋白质的磷酸化水平。比较样品之间的磷酸化水平可以提供对免疫系统状态的了解。
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| IFN-α/β Detection Assay Using Sensor Cell Lines
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Author:
Date:
2014-05-20
[Abstract] Type I interferons (IFN-α/β) play an important role in host resistance to viral infections. Signaling through the JAK-STAT pathway, IFN-α/β stimulates response elements (ISRE) in the promoters of ISG to regulate their expression (reviewed in Reference 2). This method was adapted from InvivoGen to specifically detect and quantify IFN-α/β secreted in response to virus infection. HEK-Blue™ IFN-α/β cells were generated by stably introducing the human STAT2 and IRF9 genes into HEK293 cells to obtain a fully active type I IFN signaling pathway. The activation of this pathway is made detectable by the addition of a reporter gene expressing a secreted embryonic alkaline phosphatase (SEAP) under the control of the ISG54 promoter. ISG54 is a well-known ISG activated through an ISRE-dependent ...
[摘要] I型干扰素（IFN-α/β）在宿主对病毒感染的抗性中起重要作用。 通过JAK-STAT途径的信号传导，IFN-α/β刺激ISG启动子中的反应元件（ISRE）以调节它们的表达（参见参考文献2）。 此方法改编自InvivoGen以特异性检测和量化响应于病毒感染分泌的IFN-α/β。 通过将人STAT2和IRF9基因稳定地引入HEK293细胞中以产生完全活性的I型IFN信号传导途径，产生HEK-Blue TM IFN-α/β细胞。 通过加入在ISG54启动子控制下表达分泌性胚胎碱性磷酸酶（SEAP）的报告基因，可以检测该途径的活化。 ISG54是通过I型IFN的ISRE依赖性机制激活的公知的ISG
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