H2DCFDA (DCFH-DA) belongs to the class of green fluorescent dyes and is a probe for the detection of intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm) with cell membrane permeability.

METHODS: Flow cytometry was used to detect ROS levels:
1, H2DCFDA was dissolved into 10 mM DMSO stock solution and further diluted with PBS before use.
2. Adherent cells are incubated with 5 µM H2DCFDA solution for 30 min at 37°C, protected from light, then harvested with 0.05% trypsin-EDTA solution, suspended in fresh medium and immediately analyzed by flow cytometry (488 nm). [1]
METHODS: Confocal microscopy was performed to detect ROS levels:
1. H2DCFDA was dissolved into 10 mM DMSO stock solution and further diluted with PBS before use.
2. Coverslips containing cells were placed in 5 µM H2DCFDA staining solution and incubated for 60 min at 37°C, protected from light, then washed and imaged with a confocal laser scanning microscope Leica TCS SL equipped with an argon laser. [1]

METHODS: Fluorescence microscopy was used to analyze the oxidative activity of LPS induced peritonitis in mice:
1, H2DCFDA was dissolved in 100 µL ethanol and further diluted with PBS before use.
2. C57BL/6J mice were injected intraperitoneally with LPS (0.1-1 mg/mL) to induce peritonitis. 3.5 h later, H2DCFDA (0.1-0.8 mg/ml) was injected intraperitoneally.
3. 30 min after H2DCFDA injection, the animals were killed by cervical dislocation, and the peritoneal cells were recovered by rinsing with 5 mL of ice-cold HBSS solution at pH 7.4.
4. Peritoneal cells were washed and resuspended in PBS. Macrophages were removed by adhesion method after incubation in a polystyrene dish at 37℃ for 30 min. The supernatant was recovered and approximately 20,000-25,000 leukocytes were added to microscope slides using a cytocentrifuge. The slides were then analyzed using fluorescence microscopy. [2]