Fig. 2.

Characterization of exosomes derived from Mel1 secondary melanospheres and MMP serums. (A) Transmission electron microscopy images of isolated exosomes with a saucer‐like shape limited by a lipid bilayer. EVs isolated from Mel1 secondary melanospheres culture supernatants had diameters ranging from ~ 40 to 210 nm; those isolated from MMP in had a diameter ranging from ~ 30 to 140 nm. Images show exosomes derived from an MMP at stage IV. Black arrowheads point to exosomes; (B) topography of exosomes derived from Mel1 secondary melanospheres and MMP serum observed under atomic force microscopy (AFM). Exosomes on a mica surface revealed heterogeneity in size and shape as well as forming aggregates in both 2‐dimensional 2D (left) images and 3D profiles (right). Acquisition areas were 5 × 5 µm²; (C) western blot analysis of CD9, CD63, Alix exosomal surface markers and the CD271 melanoma stem cell marker in melanosphere‐derived exosomes and Mel1 CSCs. The expression of CD9 and Alix is also shown as representative exosomal surface markers in MMP serum‐derived exosomes. IgG was used as a positive control; (D) the size distribution of exosomes obtained from Mel1 CSC and MMP serum was analysed by NTA; (E) scanning electron microscopy images of CSC‐derived exosomes aggregated (left) and individualized (right) and microanalysis of particles (down) showing the particle composition; (F) multivesicular bodies observed by electron microscopy in Mel1 CSCs. Image obtained from paraffin sections; (G) immunogold using beads coated with an anti‐CD63 antibody in exosomes derived from Mel1 secondary melanospheres cultures (left) and from a stage IV MMP serum (right).