Detection of Reactive Oxygen Species Using H2DCFDA Probe in Plant

doi:10.11983/CBB22043

Abstract

Abstract: 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.

Key words: reactive oxygen species, fluorescent probe, 2',7'-dichlorodi-hydrofluorescein diacetate, plant

Haitao Hu, Tingting Qian, Ling Yang. Detection of Reactive Oxygen Species Using H2DCFDA Probe in Plant[J]. Chinese Bulletin of Botany, 2022, 57(3): 320-326.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22043

https://www.chinbullbotany.com/EN/Y2022/V57/I3/320

Figures/Tables 2

Figure 1 Reactive oxygen species (ROS) evaluation in rice protoplasts using 2′,7′-dichlorofluorescein (DCF)-based flow cytometry (A) ROS fluorescence intensity in rice leaves under normal growth conditions; (B) ROS fluorescence intensity in rice leaves under PEG-8000 treatment conditions; (C) The relative fluorescence intensity in treatment and control groups was determined to assess the ROS content. n=3; ** indicates significant difference at P<0.01 level by Student’s t test. SSC: Side scatter

Figure 2 Confocal imaging analysis of rice leaves under normal growth (A)-(D) and PEG-8000 treatment (E)-(H) Red is the spontaneous fluorescence of chlorophyll, and green is the fluorescence of 2′,7′-dichlorofluorescein (DCF) generated by 2′,7′-dichlorodi-hydrofluorescein diacetate (H2DCFDA) oxidation. Bars=100 µm

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