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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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