A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro.
Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.
Lin
Hwang-Chi
HC
Division of Plastic Surgery, Department of Surgery, Shin-Kong Wu Ho Su Memorial Hospital, Taipei, Taiwan.
Shieh
Bin-Han
BH
Lu
Mei-Hua
MH
Chen
Jang-Yi
JY
Chang
Li-Tze
LT
Chao
Chung-Faye
CF
D001681:Biological Assay / Q000379:methods*; D018929:Cell Culture Techniques; D002460:Cell Line; D018920:Coculture Techniques; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008544:Melanocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020460:Melanosomes / Q000187:drug effects / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D000521:alpha-MSH / Q000494:pharmacology
D015842:Serine Proteinase Inhibitors; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D000521:alpha-MSH; D010088:Oxidoreductases; C064056:tyrosinase-related protein-1