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Phorbol 12-myristate 13-acetate

佛波醇12-豆蔻酸酯13-乙酸酯

Company: Sigma-Aldrich
Catalog#: P8139
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In vitro Demonstration and Quantification of Neutrophil Extracellular Trap Formation
Author:
Date:
2017-07-05
[Abstract]  In the recent decade, neutrophil extracellular traps (NETs) have been identified and confirmed as a new anti-microbial weapon of neutrophils. In this protocol, we describe easy methods to demonstrate NET formation by immunofluorescence staining of extracellular chromatin fiber with anti-DNA/Histone H1 antibody and quantification of NETs by using a non-cell-permeable DNA specific dye Sytox orange. [摘要]  近十年来,嗜中性粒细胞胞外捕获物(NETs)已被鉴定并确认为一种新的抗微生物中性粒细胞武器。 在该方案中,我们描述了通过使用抗DNA /组蛋白H1抗体的细胞外染色质纤维的免疫荧光染色和通过使用非细胞可渗透的DNA特异性染料Sytox orange来定量NETs的简便方法来证明NET形成。
【背景】嗜中性粒细胞构成循环白细胞中最大的进化保守部分。他们通过各种机制建立了针对病原体的第一道防线,包括形成嗜中性粒细胞胞外捕获物(NETs)。在此过程中,活化的嗜中性粒细胞从细胞核中排出染色质纤维。入侵的病原体然后被捕获在染色质网络内并被高度浓缩的NET缠结的抗微生物蛋白如髓过氧化物酶(MPO)和弹性蛋白酶(Brinkmann等人,2004)杀死。然而,NETs是一把双刃剑;来自过度活化的嗜中性粒细胞的无限制的NET形成也可能导致严重的组织损伤,例如通过NET的组蛋白组分的细胞毒性作用(Saffarzadeh等人,2012)。中性粒细胞过度活化并具有增强的形成NETs的能力的病理状况的一个例子是系统性红斑狼疮。狼疮患者血清中针对双链DNA及其它组分的抗体水平升高(Knight and Kaplan,2012; Yu and ...

Efficient Isolation of Influenza Specific CTLs
Author:
Date:
2016-06-20
[Abstract]  Human antigen-specific CD8+ T-cell clones are valuable tools for dissecting CD8+ T-cell responses against antigens derived from infectious agents, cancer and self antigens. Here we describe a protocol for isolating human antigen-specific CD8+ T cells. This protocol uses surface capture of IFNγ to identify antigen responsive cells that are then cloned by single-cell sorting. Here we use CD8+ T-cell responses to influenza matrix protein (MP) as an example, but this approach can be applied to any antigen specificity. [摘要]  人抗原特异性CD8 + T细胞克隆是用于解剖来自感染剂,癌症和自身抗原的抗原的CD8 + T细胞应答的有价值的工具。 在这里我们描述了用于分离人抗原特异性CD8 + T细胞的方案。 该协议使用IFNγ的表面捕获来鉴定抗原反应性细胞,然后通过单细胞分选克隆。 这里我们使用CD8 + T细胞对流感基质蛋白(MP)的反应作为例子,但这种方法可以应用于任何抗原特异性。

13C Kinetic Labeling and Extraction of Metabolites from Adherent Mammalian Cells
Author:
Date:
2015-04-20
[Abstract]  Fluctuations in metabolite levels in mammalian cells are the most direct form of readout of the cellular metabolic state. The current protocol describes a method for pulse labeling and subsequent isolation of metabolites from adherent mammalian cells. The isolated metabolites can be identified and quantified by mass-spectrometry, allowing for estimation of the rates of synthesis and removal of metabolites from the system being analyzed. [摘要]  哺乳动物细胞中代谢物水平的波动是细胞代谢状态的最直接形式的读出。 当前的协议描述了用于脉冲标记和随后从贴壁哺乳动物细胞分离代谢物的方法。 分离的代谢物可以通过质谱法鉴定和定量,允许估计代谢物从所分析的系统的合成速率和去除速率。

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