| Notch Ligand Binding Assay Using Flow Cytometry
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Author:
Date:
2017-12-05
[Abstract] Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins. Ligands expressed on one cell bind to Notch receptors on an adjacent cell to induce Notch signaling. Glycosylation of Notch receptor extracellular domain by O-fucose and ...
[摘要] Notch信号传导是进化上保守的信号传导途径，在发育过程中以及在各种组织中维持体内平衡过程中起着不可或缺的作用（Kopan，2012; Hori等人，2013）。 Notch信号的多方面作用在不同的层面上受到严格的调控。监管的最重要的方面之一是不同Notch配体与各Notch受体（NOTCH1-NOTCH4）的结合。典型的配体Delta或Serrate（在果蝇中）和Delta样（DLL1和DLL4）或Jagged（JAG1和JAG2）（在哺乳动物中）是跨膜糖蛋白。在一个细胞上表达的配体结合相邻细胞上的Notch受体以诱导Notch信号传导。通过O-岩藻糖和O-GlcNAc聚糖对Notch受体细胞外结构域的糖基化作为Notch信号强度的关键调节剂已经得到很好的确立（Stanley and Okajima，2010; Haltom and Jafar-Nejad，2015; Sawaguchi et al。 >，2017）。为了表征Notch配体与分离细胞中的Notch受体的结合，我们利用人Fc区在C端标记的Notch配体胞外结构域，并通过流式细胞术确定荧光抗Fc抗体的结合。
【背景】众所周知，细胞增殖，分化和凋亡受Notch信号调节。 ...
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| Ex vivo Human Natural Killer (NK) Cell Stimulation and Intracellular IFNγ and CD107a Cytokine Staining
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Author:
Date:
2015-06-20
[Abstract] Natural killer (NK) cells comprise 5–20% of peripheral blood mononuclear cells (PBMC) in humans. In addition to their fundamental roles in the defense against viral infections and tumor surveillance, NK cells help shape adaptive immune responses through their production of cytokines. NK cells are traditionally identified as CD3neg, CD14neg, CD19neg lymphocytes expressing CD56. Using a combination of markers that includes CD56 and CD7 greatly increases the ability to define the phenotype and function of NK cell subsets. Two key markers of NK cell function are the production of IFNγ and the release of cytotoxic granules measured by the expression of CD107a. Here we describe a method to assess IFNγ and CD107a expression in NK cells following stimulation with ...
[摘要] 自然杀伤（NK）细胞在人中包含5-20％的外周血单核细胞（PBMC）。 除了它们在防御病毒感染和肿瘤监测中的基本作用，NK细胞通过其细胞因子的产生帮助形成适应性免疫应答。 NK细胞传统上被鉴定为表达CD56的CD3阴性，CD14阳性，CD19阴性淋巴细胞。 使用包括CD56和CD7的标记物的组合极大地增加了定义NK细胞亚群的表型和功能的能力。 NK细胞功能的两个关键标记是IFNγ的产生和通过CD107a的表达测量的细胞毒性颗粒的释放。 在这里我们描述了一种方法来评估在靶细胞或细胞因子刺激后NK细胞中的IFNγ和CD107a表达。 该方法可用于评估来自广泛的研究参与者的外周血单核细胞中NK细胞的一般功能能力。
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