Hydroxyl radical production was evaluated using 3-coumarin carboxylic acid (3-CCA, J&K Chemical Co. Ltd, China) as a probe. The 3-CCA solution was prepared following the reported procedure,19 The diluted solutions, with mAuNPs concentrations of 0, 1.0, 5.0, 10.0, 20.0, and 30.0 μg/mL, were placed in Ф35-mm Petri dishes, and irradiated at 2 Gy according to the irradiation condition. After irradiation, the solutions were equally dispensed into wells of 96-well black plates, and measurements were taken at an excitation wavelength of 395 nm and an emission wavelength of 442 nm with a microplate reader (Infinite F200/M200, TECAN Co., Switzerland). The entire procedure was protected from light.

Reactive oxidative species (ROS) production was measured using the DCFH-DA assay, which is a fluorogen dye measuring hydroxyl, peroxyl, and other ROS activities within cells. B16-F10 cells were grown on glass coverslips in cell culture dishes. After co-cultivation with mAuNPs, the Au-containing medium was discarded. Cells were then co-cultured with DCFH-DA solution, which was diluted in serum-free medium for 20 minutes before irradiation. After irradiation at 2.0 Gy, the coverslips were placed on ice and quickly photographed with a fluorescence microscope. All the samples were photographed with the same microscopic parameters. The fluorescent intensities of cells were analyzed using ImageJ software. Finally, the fluorescence intensity per unit area under each treatment was calculated to ensure reliable results; at least 200 cells were randomly counted for each sample. Results are expressed as mean ± standard deviation.

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