B16F10 cells were seeded in 6-well plate at a density of 5 × 104 cells/well and incubated for 16 h. Cells were treated with 100 ng/ml α-MSH for 72 h to induce melanin synthesis and harvested by trypsin/EDTA treatment. Cells were then washed with 10 ml of 0.25 M sucrose (in 10 mM HEPES buffer) and resuspended in 10 ml of 0.25 M sucrose solution. Subsequently, after freezing with liquid nitrogen, thawing was performed at 37°C, which was repeated 10 times. After centrifugation at 1,000 g for 10 min, the supernatant was collected and further centrifuged for 45 min at 20,630 g and 4°C. Subsequently, the pellet was subjected to resuspension with DPBS and stored at –80°C before use.

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