Macrophage-Derived Exosome Isolation

Exosomes were extracted from macrophages, as reported previously [22 (link)]. Briefly, macrophages were cultured in the exosome-depleted medium for 72 h. Then, the cell medium was harvested and centrifuged at 300 g for 10 min at 4°C and the media were filtered using a 0.22 μm filter. Finally, the media was ultracentrifuged at 120000 g for 70 min at 4°C and exosomes were collected. The exosomes were resuspended in 50 μl of PBS for the following experiments. Subsequently, the isolated exosomes were determined by transmission electron microscopy (TEM), particle analyzer, and specific protein markers. Exosomes were fixed with 1% buffered glutaraldehyde for 10 min. The 20 μL of exosomes were added to formvar carbon-coated 300-mesh copper electron microscopy grids (Agar Scientific Ltd., Stansted, UK) and allowed to stand for 5 min. Then 2% uranyl oxalate was added to counterstain exosomes at room temperature. After the grids were washed three times with PBS, the exosomes were photographed using TEM (Hitachi H7500 TEM, Tokyo, Japan). The exosome particle size analysis was conducted by nanoparticle tracking analysis (NTA) with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The CD63 was applied to identify exosomes via western blot analysis.