fig4

Figure 4. Characterization of Nycodenz DG-purified MEG-01 lEV and sEV. MEG-01-derived lEVs and sEVs were isolated from conditioned medium by differential ultracentrifugation and further purified by Nycodenz DG centrifugation to separate EVs from non-EV contaminants; (A) Total protein staining of density gradient fractions of 10,000 × g (lEV) pellet; (B) Total protein staining of density gradient fractions of 100,000 × g (sEV) pellet; (C) Western blot for Annexin A1, a lEV marker, shows enrichment of lEVs in the 4th-7th fractions; (D) Western blots for classical sEV markers CD63 and CD81 show enrichment in the 4th-7th fractions of the sEV gradient; (E) TEM images of DG-purified lEVs and sEVs reveal vesicular structures with EV-characteristic morphology. Scale bars: 100 nm; (F) Mode size distribution of purified lEVs and sEVs determined by NTA. Representative total protein staining (A and B), immunoblots (C and D), and TEM images (E) from one of three independent biological experiments with similar results are shown. Quantitative data in panel F are presented as mean ± SD from three independent biological experiments. Statistical significance was determined using an unpaired two-tailed Student’s t-test; ^***P < 0.001. DG: Density gradient; EV: extracellular vesicle; lEV: large extracellular vesicle; NTA: nanoparticle tracking analysis; SD: standard deviation; sEV: small extracellular vesicle; TEM: transmission electron microscopy.