Biological characteristics of mouse skin melanocytes

• The proliferation of melanocytes from passages 4, 7, 10 and 13 was determined using RTCA iCELLigence Analyzer.
• Quantitative real time PCR analysis of expression of coat color genes.
• Immunoreactivity of the melanocyte specific markers.

The objective of this research was to evaluate the optimal passage number according to the biological characteristics of mouse skin melanocytes from different passages. Skin punch biopsies harvested from the dorsal region of 2-day old mice were used to establish melanocyte cultures. The cells from passage 4, 7, 10 and 13 were collected and evaluated for their melanogenic activity. Histochemical staining for tyrosinase (TYR) activity and immunostaining for the melanocyte specific markers including S-100 antigen, TYR, tyrosinase related protein 1 (TYRP1), tyrosinase related protein 2 (TYRP2) and micropthalmia associated transcription factor (MITF) confirmed purity and melanogenic capacity of melanocytes from different passages, with better melanogenic activity of passage 10 and 13 cells being observed. Treatment of passage 13 melanocytes with α-melanocyte stimulating hormone (α-MSH) showed increased expression of MITF, TYR and TYRP2 mRNA. However, considering the TYR mRNA dramatically high expression which is the characteristics of melanoma cells, melanocytes from passage 10 was the optimal passage number for the further research. Our results demonstrate culture of pure populations of mouse melanocytes to at least 10 passages and illustrate the potential utility of passage 10 cells for studies of intrinsic and extrinsic regulation of genes controlling pigmentation and coat color in mouse.