Rabbit Melanocyte Culture and Manipulation

Melanocyte culture was established using the method described by Chen et al. [22 (link)]. An adult black rabbit was used to isolate the melanocytes. A tissue sample of approximately 1.5 cm × 1.5 cm was excised from the dorsal skin of the rabbit. Rabbit melanocytes were cultured in Melanocyte Medium-2 (ScienCell, San Diego, CA, USA) supplemented with 1% PMA-free melanocyte growth supplement, 0.5% fetal bovine serum (FBS), and 0.5% penicillin/streptomycin solution (ScienCell, San Diego, CA, USA). Melanocytes were transferred to 24-well plates and incubated at 37 °C with 5% CO₂. When the culture was 70–90% confluent, overexpression and knockdown experiments were performed using 1 μg GNAI2 plasmid and 1 μL siRNAs (Table 2), respectively. Both were diluted in 25 μL Opti-MEM™ medium (Gibco, Carlsbad, CA, USA) and 2 μL diluted Lipofectamine™ 3000 (Lipofectamine™ 3000 diluted in 25 μL Opti-MEM™ medium) was added to both the mixtures. These mixtures were incubated for 10-15 min at room temperature and were then added to cultured melanocytes separately, and the cells were incubated at 37 °C with 5% CO₂ for 48 h.