2',7'-Dichlorofluorescein diacetate (Diacetyldichlorofluorescein), a cell-permeable fluorogenic probe, is useful for the detection of nitric oxide (NO) and reactive oxygen species (ROS) and for the determination of the degree of overall oxidative stress.

I. Detection of Reactive Oxygen Species (ROS)
1. Material preparation
1) DCFH-DA probe solution: Prepare DCFH-DA stock solution (1–10 mM), dissolve it in anhydrous DMSO, and store it away from light.
2) Cell sample: Living cells or tissue samples to be tested.
3) PBS buffer: Used to wash cells, pH 7.4.
2. Experimental steps
1) Cell treatment: Collect the cells to be tested, wash them with PBS 2-3 times to remove interferences in the culture medium.
2) Staining: Dilute DCFH-DA to 0.1–20 µM, add it to the cell culture medium, incubate at 37°C for 30 minutes, and protect from light.
3) Washing: After incubation, wash the cells with PBS buffer to remove DCFH-DA that has not entered the cells.
4) Detect fluorescence: Use a fluorescence microscope or flow cytometer to detect the fluorescence signal.
Excitation wavelength: 488 nm
Emission wavelength: 525 nm
II. Detection of Nitric Oxide (NO)
DCFH-DA detects the presence of NO through an indirect mechanism, depending on the secondary products generated by NO (such as peroxynitrite).
The experimental steps are similar to ROS detection and are suitable for indirect assessment of changes in NO content.
III. Assessment of total oxidative stress
1) Compare the fluorescence signal with the standard curve to quantitatively determine the level of ROS or NO.
2) Use blank groups (cells not treated with DCFH-DA) and positive controls (such as H2O2 or NO donor treatment groups) to verify the reliability of the experimental results.