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PBS, pH 7.4

PBS,pH 7.4

Company: Thermo Fisher Scientific
Catalog#: 10010023
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Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
Author:
Date:
2018-07-20
[Abstract]  After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow ... [摘要]  在识别细胞外细菌脂多糖(LPS)后,Toll样受体4(TLR4)-CD14信号传导复合物启动两种不同的信号传导途径 - 一种来自质膜,另一种来自信号传导内体(Kagan 等。,2008)。因此,了解TLR4信号转导的早期阶段需要一种稳健且定量的方法来测量LPS触发的TLR4和CD14受体内吞作用,这是LPS检测中最早发生的事件之一。在这里,我们描述了一种基于流式细胞术的方法,我们最近用它来研究离子通道TRPM7在TLR4内吞作用中的作用(Schappe et al。,2018)。该测定依赖于用LPS刺激细胞并使用流式细胞术在不同时间点测量TLR4(或CD14)的细胞表面水平。尽管我们详细描述了来自鼠骨髓来源的巨噬细胞的TLR4和CD14的方法,但它可以很容易地适应于在各种其他信号传导环境中评估受体内吞作用。

【背景】先天免疫细胞,包括巨噬细胞和树突细胞,使用各种模式识别受体(PRR)来调查其环境中的危险和病原体相关分子模式。来自各种亚细胞区室的PRR的贩运和信号传导实现了更广泛的免疫监视,并且已成为先天免疫的重要设计原则(Brubaker et al。,2015)。细菌内毒素LPS的检测高度依赖于TLR4及其共同受体CD14。 TLR4复合物的内吞作用需要CD14,并且对于LPS诱导的巨噬细胞活化是必需的(Zanoni 等人,2011; Tan ...

Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry
Author:
Date:
2018-07-20
[Abstract]  Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD+) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD+ is a key factor for the development of age-associated metabolic decline. Increased NAD+ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging.

Given the importance of monitoring cellular NAD+ and ...
[摘要]  目前关于代谢功能障碍和虚弱的年龄相关发展的研究每天都有更多的证据。众所周知,随着衰老的进展,烟酰胺腺嘌呤二核苷酸(NAD + )水平在预期的生理过程中降低。最近的研究表明,NAD + 的减少是与年龄相关的代谢衰退发展的关键因素。增加NAD + 水平体内导致激活寿命和健康跨度相关因素。此外,它改善了老化的几个生理和代谢参数,包括自然和加速老化的小鼠模型中的肌肉功能,运动能力,葡萄糖耐量和心脏功能。

鉴于监测细胞NAD + 和NADH水平的重要性,有一个值得信赖的方法是至关重要的。该方案的目的是在有效且广泛适用的测定中描述来自组织和细胞的NAD + 和NADH提取以及其图形和定量分析。

【背景】氧化烟酰胺腺嘌呤二核苷酸和还原型烟酰胺腺嘌呤二核苷酸(NAD + 和NADH)是重要的生物辅助因子,它们在几种合成代谢和分解代谢功能中提供和接受电子。它们参与诸如糖酵解,三羧酸循环和氧化磷酸化的反应。此外,它还作为DNA损伤修复中涉及的几种酶的底物,例如sirtuins和poly(ADP-核糖)聚合酶(PARP)(Imai和Guarente,2014; Verdin,2015; Yoshino et al。 ,2018)。

NAD + ...

Measuring CD38 Hydrolase and Cyclase Activities: 1,N6-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) Fluorescence-based Methods
Author:
Date:
2018-07-20
[Abstract]  CD38 is a multifunctional enzyme involved in calcium signaling and Nicotinamide Adenine Dinucleotide (NAD+) metabolism. Through its major activity, the hydrolysis of NAD+, CD38 helps maintain the appropriate levels of this molecule for all NAD+-dependent metabolic processes to occur. Due to current advances and studies relating NAD+ decline and the development of multiple age-related conditions and diseases, CD38 gained importance in both basic science and clinical settings. The discovery and development of strategies to modulate its function and, possibly, treat diseases and improve health span put CD38 under the spotlights. Therefore, a consistent and reliable method to measure its activity and explore its use in medicine is required. We ... [摘要]  CD38是参与钙信号传导和烟酰胺腺嘌呤二核苷酸(NAD + )代谢的多功能酶。通过其主要活性,NAD + 的水解,CD38有助于维持该分子的适当水平,从而发生所有NAD + 依赖性代谢过程。由于目前有关NAD + 下降和多种年龄相关疾病和疾病发展的研究和进展,CD38在基础科学和临床环境中都具有重要意义。调整其功能,可能治疗疾病和改善健康状况的战略的发现和发展使CD38成为焦点。因此,需要一种一致且可靠的方法来测量其活性并探索其在医学中的用途。我们在这里描述了我们的小组如何测量CD38的水解酶和环化酶活性的方法,利用基于荧光的酶测定法在平板阅读器中使用1,N 6 -Ethenonicotinamide Adenine Dinucleotide(ε-NAD)进行)和烟酰胺鸟嘌呤二核苷酸(NGD)分别作为底物。

【背景】目前关于代谢功能障碍和虚弱的年龄相关发展的研究每天都有更多的证据。众所周知,随着老化的进行,NAD + 水平在预期过程中降低。最近的研究表明,烟酰胺腺嘌呤二核苷酸(NAD + )的减少与年龄相关的代谢下降有关(Massudi et al。,2012)。增加NAD + 水平体内,导致延长寿命和健康跨度相关因素的活化,并改善衰老的几个生理和代谢参数(Camacho-Pereira ...

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