DHE is one of the most commonly used superoxide anion (O2-) fluorescent probes for the detection of membrane permeability. It can be directly used to detect oxidative activity of living cells, including ROS (reactive oxygen species) and superoxide detection.

After DHE enters living cells, it dehydrogenates under the action of superoxide anion and oxidizes to form Ethidium. Ethidium binds to DNA to produce red fluorescence. When the level of superoxide anion in the cell is higher, more ethidium is produced, and the red fluorescence is stronger, and vice versa. DHE itself fluoresces blue (λEx /λEm: 355/420nm) until it is oxidized to Ethidium, and then embedded in DNA, so that the nucleus is bright red fluorescence (λEx /λEm: 518/605 nm), which can be detected by fluorescence microscopy and flow cytometry.

DHE is a permeable blue fluorescent probe, a peroxide indicator, which can be used to detect intracellular superoxide radical anion, and can effectively detect reactive oxygen species. The dye enters the cell freely and dehydrogenates to become Ethidium. DHE can be used directly to label living cells. After ingestion by living cells, DHE can dehydrogenate under the action of superoxide anions in cells to produce Ethidium. Ethidium can bind to RNA or DNA to produce red fluorescence. When the level of superoxide anion in the cell is higher, more Ethidium is produced, and the red fluorescence is stronger, and vice versa. DHE itself is blue fluorescence, the maximum excitation wavelength is 370nm, the maximum emission wavelength is 420nm, after dehydrogenation and RNA or DNA combination to produce red fluorescence, the maximum excitation wavelength is 300nm, the maximum emission wavelength is 610nm, the actual observation can also use 535nm as the excitation wavelength.

In general, when used for intracellular superoxide anion detection, the common concentration of DHE is 1-10μM.

Operation method (for reference only)

Dyeing method:

(1) The probe solution can be diluted to the required concentration in fresh culture solution, buffer salt solution or tissue perfusion solution, and the staining solution can replace the cell culture solution or perfusion solution; The probe can also be added directly into the cell incubation or perfusion solution to the desired concentration.

(2) Depending on the ROS content of cells, the final concentration of DHE can be selected in the range of 1 μM~100 μM, and the incubation time can be selected for 10~90 min. Incubation can be carried out at 37 ° C or room temperature, requiring protection from light.

(3) After incubation, clean the cells or tissues with fresh solution.

Fluorescence microscope:

(1) The adherent growth cells or living tissues can be observed directly under a fluorescence microscope; For suspended growth cells, 25-50 μL cell suspension was dropped onto a microscopic slide and then covered with a cover slide.

(2) Under fluorescence microscopy, red emission images of cells were observed and photographed using blue or green light excitation, and ROS-positive cells were dyed red throughout the nuclear region; When excited by ultraviolet light, the unoxidized DHE in the cytoplasm can emit blue fluorescence.

Flow cytometry:

(1) The adherent growth cells were digested with pancreatic enzymes to prepare single-cell suspension; For suspended growth cells, cells are collected directly. The cells were suspended with 0.5 to 1 mL cold PBS (50,000 to 100,000).

(2) The emission above 590 nm to 610 nm was measured by excitation at 480~535 nm, and the cells could be divided into two subgroups: ROS negative cells had very low fluorescence intensity, and ROS positive cells had strong red fluorescence.

Matters needing attention

1. DHE is easily oxidized in light and air, and should be kept away from light.

2. For different cells and tissues, appropriate incubation time and concentration should be selected to observe the changes of ROS.

Examples of using this product（for reference only）

ROS :

ROS production in root tips of the treated lettuce seedlings was estimated by staining with DHE (Solarbio, Beijing, China) according to Yamamoto et al. Lettuce roots were soaked in the dye solution (DHE, 10 mM; acetone, 0.01%; CaCl2, 100 mM; pH 4.75) with gentle shake for 10 min at 25℃ in the dark. After washing with 100 mM CaCl2 for 20 min, the fluorescence was observed and photographed using a fluorescence microscope with excitation 488 nm and emission > 510 nm. At least 15 roots per treatment were analyzed and the representative results were shown in the figures.

Reference：

Yan ZQ, Tan J, Guo K, Yao LG. Phytotoxic mechanism of allelochemical liquiritin on root growth of lettuce seedlings. Plant Signal Behav. 2020 Oct 2;15(10):1795581. doi: 10.1080/15592324.2020.1795581. Epub 2020 Jul 21. PMID: 32693669; PMCID: PMC8550531.

ROS Measurement:

For DHE staining, flies were anesthetized with CO2 and dissected in cold PBS under a dissecting microscope. In ROS staining assay, 30 guts and 8 brains were assayed. The dissected brains and guts were incubated at 25 ℃ with 60 mM DHE for 25 and 15 min, respectively. Next, we washed the samples for 3×5 min in PBS at room temperature. Before mounting, the samples were incubated at 25 ℃ with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min and then washed three times for 5 min with PBS at room temperature. All steps were performed on a shaker in the dark.

Reference：

Wei JJ, Li XJ, Liu W, Chai XJ, Zhu XY, Sun PH, Liu F, Zhao YK, Huang JL, Liu YF, Zhao ST. Eucommia Polysaccharides Ameliorate Aging-Associated Gut Dysbiosis: A Potential Mechanism for Life Extension in Drosophila. Int J Mol Sci. 2023 Mar 20;24(6):5881. doi: 10.3390/ijms24065881. PMID: 36982954; PMCID: PMC10054339.