Different approaches for assaying melanosome transfer

• Published in: Pigment cell research Vol. 18; no. 5; pp. 370 - 381
• Main Authors: Berens, Werner, Van Den Bossche, Karolien, Yoon, Tae-Jin, Westbroek, Wendy, Valencia, Julio C., Out, Coby J., Marie Naeyaert, Jean, Hearing, Vincent J., Lambert, Jo
• Format: Journal Article
• Language: English
• Published: Oxford, UK Munksgaard International Publishers 01.10.2005
• Subjects: Animals; Antigens, Neoplasm; Carbon Radioisotopes - analysis; Cell Line; Cell Separation; Coculture Techniques; Flow Cytometry; Fluoresceins; GFP; Green Fluorescent Proteins - genetics; Humans; Hydrogen-Ion Concentration; Immunomagnetic Separation; keratinocyte; Keratinocytes - metabolism; MART-1 Antigen; melanin; Melanins - analysis; Melanins - chemistry; Melanins - metabolism; melanocyte; Melanocytes - metabolism; Melanosomes - metabolism; Mice; Microscopy, Confocal; Monophenol Monooxygenase - genetics; Monophenol Monooxygenase - metabolism; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Organometallic Compounds; Pigmentation; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Staining and Labeling - methods; thiouracil; Thiouracil - chemistry; Transfection; transfer; Trypsin
• ISSN: 0893-5785; 1600-0749
• DOI: 10.1111/j.1600-0749.2005.00263.x

Summary Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP‐labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C‐thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co‐culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer.