| 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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| Minimal Bactericidal Concentration for Biofilms (MBC-B)
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Author:
Date:
2014-05-05
[Abstract] A biofilm is a multicellular consortium of surface associated microbes surrounded by a hydrated, extracellular polymer matrix. The biofilm matrix plays a critical role in preventing desiccation, acquiring nutrients, and provides community protection from environmental assaults. Importantly, biofilms are significantly more resistant to antimicrobials relative to their free-swimming counterparts. The level of antimicrobial tolerance is influenced by a number of factors, including genetic/adaptive resistance mechanisms, stage of biofilm development, and pharmacokinetics of the antibiotic. Here, we describe an in vitro microtiter-based assay to quantify the minimal bactericidal concentration for biofilms (MBC-B) for short exposure times (2 h). This exposure period is significantly ...
[摘要] 生物膜是由水合的细胞外聚合物基质包围的表面相关微生物的多细胞聚生体。生物膜基质在防止干燥，获得营养物质和提供社区保护免受环境攻击中起关键作用。重要的是，相对于其自由游泳的对应物，生物膜显着地更抗抗微生物剂。抗微生物耐受性的水平受许多因素影响，包括遗传/适应性抗性机制，生物膜发育阶段和抗生素的药代动力学。在这里，我们描述了一种基于体外微量滴定的测定法，用于定量短曝光时间（2小时）的生物膜（MBC-B）的最小杀菌浓度。该暴露时间显着短于传统方案中描述的标准过夜和24小时治疗。开发该测定法以接近抗生素在其被代谢，被宿主蛋白螯合或消化之前在一次性治疗期间可用的时间。
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