| Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells
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Author:
Date:
2020-09-05
[Abstract] Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation of immune responses. The ROS-sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is an inexpensive and well-established tool for measuring intracellular ROS levels. However, it needs to be carefully controlled to be able to draw firm conclusions on the nature of ROS species produced and the cellular source of ROS generation such as the enzyme complex NADPH-oxidase 2 (NOX-2). In this protocol, a robust method to ...
[摘要] [摘要 ] 取决于其浓度和细胞来源，生物体中活性氧（ROS）的产生具有多种功能。虽然在氧化性爆发期间使用高浓度来对抗病原体，但低至中等量的ROS充当信号分子，对几种生理过程（例如免疫应答的调节）很重要。ROS敏感染料2'，7'-二氯二氢荧光素二乙酸酯（H 2 DCFDA）是一种用于测量细胞内ROS水平的廉价且行之有效的工具。但是，需要仔细控制它，以便能够对产生的ROS种类的性质和ROS产生的细胞来源（如酶复合物NADPH-氧化酶2（NOX-2））得出可靠的结论。在该协议中，使用H 2 DCFDA 确定低细胞内ROS产生的可靠方法已通过几种ROS特异性抑制剂和NOX-2特异性抑制剂的验证。在用感兴趣的ROS-诱导剂处理之前，用抑制剂或对照物质处理细胞。仅在治疗计划的最后30分钟内添加H 2 DCFDA。为了终止其转化，我们使用了ROS特异性抑制剂，直到通过FITC通道（例如：488 nm / Em ：519 nm）通过流式细胞仪进行分析。总之，该协议允许检测细胞系和原代免疫细胞（例如，分别为Mono Mac 6细胞和骨髓来源的树突状细胞）中与信号相关的细胞内ROS的产生。结合使用这种方法和特定的抑制剂，我们甚至能够验证NOX-2产生的与免疫调节信号相关的极少量的ROS。
[背景 ] ...
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| Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes
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Author:
Date:
2020-04-05
[Abstract] Glucocerebrosidase (GCase) is an important enzyme for the metabolism of glycolipids. GCase enzyme deficiency is implicated in human disease and the efficient measurement of GCase activity is important for evaluating the efficacy of therapeutics targeting this enzyme. Existing approaches to measure GCase activity include whole blood mass spectrometry-based assays, where an internal standard is used to measure the accumulation of ceramide following metabolism of the synthetic substrate C12-glucocerebroside, and the utilisation of fluorescent probes that bind active GCase and/or release fluorescent metabolites upon cleavage by GCase. Here, we describe the application of a fluorescence-activated cell sorter-based assay to efficiently quantitate GCase enzyme activity in the monocyte population ...
[摘要]
[摘要 ] 葡萄糖脑苷脂酶（GCase ）是糖脂代谢的重要酶。GCase 酶缺乏症与人类疾病有关，GCase 活性的有效测量对于评估靶向该酶的治疗剂的功效至关重要。现有的测量GCase 活性的方法包括基于全血质谱的分析，其中使用内标测量合成底物C12-葡萄糖脑苷代谢后神经酰胺的积累，以及利用结合活性GCase 和// 的荧光探针的利用。或通过GCase 裂解后释放荧光代谢物 。在这里，我们描述了基于荧光激活细胞分选术的测定方法的应用，以有效地定量人类外周血单核细胞单核细胞群中的GCase 酶活性。细胞渗透性的GCase 衬底5-（Pentafluorobenzoylamino ）荧光素二-β-D- 吡喃葡萄糖苷（PFB- FDGlu ）提供了一个用于测量的GCase 活性，由此酶裂解产生的绿色荧光PFB-F染料，在FL-检测流式细胞仪的1个通道。使用溶酶体GCase 活性抑制剂，conduritol B-环氧，以确保特异性。该协议为测量活的单个细胞中的GCase 活性提供了一种有利的方法。
[背景 ] 葡萄糖脑苷脂酶（GCase ），由 GBA1基因是一种溶酶体水解酶，可将葡糖神经酰胺转化为葡萄糖和神经酰胺。GBA1 ...
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| Analyzing the Quenchable Iron Pool in Murine Macrophages by Flow Cytometry
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Author:
Date:
2020-03-20
[Abstract] Tissue-resident macrophages are pivotal for a tightly-regulated iron metabolism at a cellular and systemic level, since subtle iron alterations increase the susceptibility for microbial infections or drive multiple diseases. However, research on cellular iron homeostasis in macrophages remains challenging due to the limited amount of available methods using radioactive 59Fe isotopes or strong iron chelators, which might be inapplicable in certain experimental settings. This protocol describes the analysis of the quenchable iron pool (QIP) in macrophages by loading these cells with exogenous iron-complexes. Thereby, the cytoplasmic iron pool can be determined, since the iron uptake ability of macrophages inversely correlates with intracellular iron levels. Thus, this assay ...
[摘要] [摘要 ] 驻留在组织中的巨噬细胞对于在细胞和全身水平上严格调节铁代谢至关重要，因为细微的铁改变会增加对微生物感染的敏感性或引发多种疾病。然而，由于使用放射性59 Fe同位素或强铁螯合剂的可用方法数量有限，因此巨噬细胞中细胞铁稳态的研究仍然具有挑战性，这在某些实验环境中可能不适用。该协议 描述了通过向这些细胞加载外源铁配合物来分析巨噬细胞中的可淬灭铁池（QIP）。因此，由于巨噬细胞的铁摄取能力与细胞内铁水平成反比，因此可以确定细胞质的铁库。因此，该测定法能够准确分析细胞质铁通量的微小变化，并且几乎适用于所有实验室环境。另外，该方案还可以用于体外和体内的其他免疫细胞类型。
[背景 ] 由于用于全身铁代谢它们的中心调节功能，在多种组织中的巨噬细胞中迅速地改变在不同的刺激的遭遇它们的细胞内的铁水平的微生物感染和疾病（Nairz 等人，2017） 。值得注意的是，大多数细胞中的铁离子被细胞质中的主要铁存储蛋白铁蛋白结合或定位于线粒体或溶酶体等区室（Ma 等，2015 ; Soares和Hamza，2016 ...
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