| 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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| HIV-1 Fusion Assay
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Author:
Date:
2014-08-20
[Abstract] The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase–Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).
[摘要] HIV-1融合测定测量HIV-1生命周期中直至并包括病毒融合的所有步骤。 它依赖于将β-内酰胺酶-Vpr（BlaM-Vpr）蛋白嵌合体并入病毒体中，并且随后通过融合将该嵌合体转移到靶细胞中（图1）。 通过CCF2（一种β-内酰胺酶的荧光染料底物）的酶裂解来监测转移，将其装载到靶细胞中。 β-内酰胺酶在CCF2中的β-内酰胺环的切割将染料的荧光发射光谱从绿色（520nm）改变为蓝色（447nm）。 这种变化反映了病毒体融合，并且可以通过流式细胞术检测（图2）。
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