FIGURE 1.
Exosome release in acidic and buffered pH conditions. A, exosomes (100 μg) isolated from Mel1 cells culture medium were NHS-biotin-labeled and loaded on a 10–60% continuous sucrose gradient. Eleven fractions were analyzed by Western blotting with horseradish peroxidase-streptavidin. Results indicate a molecules profile with molecular mass ranging between 97 and 21 kDa enriched in fractions 3–6 corresponding to density 1.11–1.17 g/ml. These fractions were also found positive for exosome markers such as Lamp-2, CD81, and Rab 5B by Western blotting. B, Mel1 cells (3 × 106 cell/ml) were cultured in buffered (pH 7.4) or acidic (pH 6.0) media. At the indicated days exosomes were isolated, quantified by protein assay, and normalized on 1 × 106 viable cells. Points, means (n = 4); bars, S.D. *, p < 0.05. C, Western blotting of Lamp-2 and Rab 5B in exoMel1ac and exoMel1 released from parental cells at 2, 3, and 4 days. Note that Lamp-2 and Rab 5B expressions increased with time in the acidic exosomes. A representative Western blotting of three independent experiments is shown.