Serum exosomes were isolated using ultracentrifugation as previously described with modifications [14 (link)]. Briefly, the serum was obtained by centrifugation at 3000 × g for 15 min to remove the cell debris and platelets. Microvesicles were pelleted at 10,000 × g for 30 min, after which the exosomes were further purified from the supernatant by ultracentrifugation at 100,000 × g for 60 min. Following isolation, the exosomes were diluted in 100 µL of filtered PBS and stored at − 80 °C.
Exosomes were dissolved in lysis buffer and quantified using a BCA analysis kit (Thermo Fisher Scientific, USA). Western blotting was used to detect exosomal markers, including TSG101, HSP70, CD63 and CD9. Exosomes were fixed in 2.5% glutaraldehyde at 4 °C, dehydrated with gradient alcohol and embedded in epoxy resin. Sections were stained with uranyl acetate and citrate acid lead. The images were captured under a transmission electron microscope (JEM-1010, Japan). For nanoparticle tracking analysis, samples were loaded into the sample chamber of an NS500 unit (NanoSight, UK), and five 1-min videos of each sample were recorded. The data analysis was performed with NTA 2.3 software, and the size and concentration of the particles were calculated.
Exosomes were dissolved in lysis buffer and quantified using a BCA analysis kit (Thermo Fisher Scientific, USA). Western blotting was used to detect exosomal markers, including TSG101, HSP70, CD63 and CD9. Exosomes were fixed in 2.5% glutaraldehyde at 4 °C, dehydrated with gradient alcohol and embedded in epoxy resin. Sections were stained with uranyl acetate and citrate acid lead. The images were captured under a transmission electron microscope (JEM-1010, Japan). For nanoparticle tracking analysis, samples were loaded into the sample chamber of an NS500 unit (NanoSight, UK), and five 1-min videos of each sample were recorded. The data analysis was performed with NTA 2.3 software, and the size and concentration of the particles were calculated.
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