The cells were co-cultured with AuNPs-containing medium at Au concentrations of 5.0, 10.0, 20.0, 40.0, 60.0 μg/mL for 24 h, respectively, and the medium was discarded. B16-F10 cells were then washed with PBS and harvested using trypsin (Hyclone, US). The cell number was counted using a particle counter (Z1, Beckman Coulter Co., US). The cell suspension was centrifuged for 20 min at 12,000 rpm. The collected cells are ruptured using ultrapure water using osmotic pressure. The cells are also broken more thoroughly with ultrasound to ensure that the Au inside the cells is completely released. The pellets were dissolved by aqua regia, and then the solution was diluted with ultrapure water. The concentration of Au was determined using ICP-AES, and the results were normalized by cell numbers. The concentration of mAuNPs used in all subsequent cell experiments was the same as that used in the co-culture concentrations.

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