Efficacy of quantifying melanosome transfer with flow cytometry in a human melanocyte-HaCaT keratinocyte co-culture system in vitro

Abstract

In this study, we describe a simple, specific, reproducible and quantitative assay system to assess melanosome transfer. We first established a co-culture model of normal human epidermal melanocytes and HaCaT keratinocytes. The cells were co-cultured for 72 h in a serum-free keratinocyte growth media and double labelled with Fluorescein isothiocyanate (FITC)-conjugated antibody against the melanosome-specific protein gp100, and with Phycoerythrin (PE)-conjugated antibody against the keratinocyte-specific marker cytokeratin. Then, the cells were examined using co-focal microscope and flow cytometry. The increased melanosome transfer from melanocytes to HaCaT keratinocytes was observed in a time-dependent manner. To verify the accessibility of this method, two known melanosome transfer inhibitors and two known melanosome transfer stimulators were applied. Consistent with previous investigation, soybean trypsin inhibitor (STI), niacinamide inhibited melanosome transfer, α-melanocyte stimulating hormone (α-MSH) and keratinocyte growth factor (KGF) increased melanosome transfer, respectively, in a dose-dependent manner. The model used in this study could thus represent a rapid and reliable tool to identify modulators of human melanosome transfer. © 2009 John Wiley & Sons A/S.