基于H2DCFDA荧光探针的植物活性氧检测方法

活性氧(reactive oxygen species, ROS)是植物体内的一把“双刃剑”。ROS作为信号分子在植物生命活动中发挥关键作用,但ROS过量积累会对生物大分子造成氧化损伤。准确测定ROS含量对于评估植物细胞内的氧化还原状态至关重要。由于植物体内ROS各组分半衰期短且反应活性强,定性定量检测较为困难。因此,选择合适的检测方法以提高检测的时空准确性非常重要。目前,荧光分析法因其具有灵敏度高、选择性好、检出限低和直观性强等优点,受到研究人员的广泛关注。该文详细描述基于流式细胞仪和激光共聚焦显微镜,利用2′,7′-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针检测水稻(Oryzasativa)体内ROS水平和时空分布的操作流程及注意事项。该技术也可用于直接检测拟南芥(Arabidopsis thaliana)、玉米(Zea mays)和大豆(Glycine max)等模式植物组织中ROS的水平和分布。

Detection of Reactive Oxygen Species Using H2DCFDA Probe in Plant

Reactive oxygen species (ROS) are a ‘double-edged sword’ in plants. On the one hand, ROS, as a signal molecule, plays pivotal roles in many aspects of life activities; on the other hand, excessive accumulation of ROS can cause oxidative damage to biological macromolecules. Accurate detection of ROS is essential to assess its intracellular redox status. Due to the characteristics of short half-life and strong reactivity of ROS components, their qualitative and quantitative analysis are difficult. It is critical to select the appropriate detection method and improve the spatiotemporal accuracy of detection for research in plant sciences and in other fields. At present, fluorescent probe analysis has attracted the attention of researchers because of its advantages of high sensitivity, good selectivity, low detection limit and strong intuition. This article introduces the detailed operation protocol and attentions for ROS detection using 2′,7′-dichlorodi-hydrofluorescein diacetate (H2DCFDA) fluorescent probe based on flow cytometry and confocal microscope. These methods can be used to detect ROS levels and distribution in model plant tissues, including Oryza sativa, Arabidopsis thaliana, Zea mays and Glycine max.