Dcfh da

The F9 cells were treated with AgNPs or AgNO₃ for 24 h. ROS were measured according to a previous method based on the intracellular peroxide-dependent oxidation of DCFH-DA (Molecular Probes) to form the fluorescent compound 2′,7′-dichlorofluorescein (DCF).7 (link),11 (link) Cells were seeded onto 24-well plates at a density of 5×10⁴ cells per well and cultured for 24 h. After washing twice with PBS, fresh medium containing AgNPs or AgNO₃ was added, and the cells were incubated for 3 h. For the control, 20 μM DCFH-DA was added to the cells and incubated for a further 30 min at 37°C. The cells were then rinsed with PBS, and 2 mL of PBS was added to each well and the fluorescence intensity was determined using a spectrofluorometer (Gemini EM) with excitation at 485 nm and emission at 530 nm. DCFH-DA (20 μM) was then added, and the cells were incubated for 30 min at 37°C before measuring the changes in DCF fluorescence as described.

Levels of intracellular ROS were assessed by use of a reactive oxygen species assay kit (Beyotime, Nanjing, China). dichlorofluoresceindiacetate(DCFH-DA, Molecular Probes,Beyotime) was used. This reagent enters the cells and reacts with ROS, producing the fluorophoredichlorofluorescein (DCF). Briefly, cell cycle phase was either kept untreated or exposed to 2 h 43°C, and further incubated for different times, then about 3 × 10⁵ were harvested, washed with serum free DMEM culture medium and the cells were stained with10 μM dichlorofluoresceindiacetate (DCFH-DA, Molecular Probes,Beyotime) for 30 min at 37°C in the darkness. Then the cells were harvested, washed and resuspended in serum-free DMEM culture medium for three times. The cells' fluorescence intensity was determined using flow cytometer52 (link). 200 μM H₂O₂ –treated HUVEC cells were as a positive control.