Quantifying Melanocyte Melanosome Stages

Control- and peptide-treated MNT-1 cells grown on coverslips for 3 days—in media containing or not 10 µM peptides (media were renewed every day)—were fixed with 2.5% glutaraldehyde/0.1 M cacodylate buffer, pH 7.2, post-fixed with 1% OsO₄ supplemented with 1.5% potassium ferrocyanure, dehydrated in ethanol, and embedded in Epon. Ultrathin sectioning was performed with an ultramicrotome (UCT, Leica, Wetzlar, Germany). Sections were counter-stained with uranyl acetate and lead citrate and electron micrographs were acquired under electron microscopes (Philips CM120 or Tecnai Spirit G2; FEI, Eindhoven, The Netherlands) equipped with a numeric camera (Keen View; Soft Imaging System) or 4k CCD camera (Quemesa, Olympus, Muenster, Germany). Melanosome stages from I to IV were quantified based on their morphology and melanin content (see Table 2 and [3 (link)]). Two independent quantifications by two different authors were performed on more than 100 structures. Results presented in Figure 2c: Stage I: H₂O, 4 ± 3%; QK-5, 5 ± 3%; Stage II: H₂O, 11 ± 7%; QK-5, 17 ± 11%; Stage III: H₂O, 31 ± 8%; QK-5, 52 ± 8%; Stage IV: H₂O, 55 ± 11%; QK-5, 26 ± 16%.