Exosome Isolation and Characterization from Macrophages

The exosomes secreted by macrophages were isolated using gradient centrifugation [29 (link)]. Briefly, macrophages were cultured for 48 hours in exosome-free medium. Then, the macrophage culture medium was collected and centrifuged at 1000 × g for 10 min, 3000 × g for 30 min, 6000 × g for 40 min, and 12,000 × g for 2 hours to remove the cell debris. The supernatant was collected and centrifuged at 100,000 × g for 4 h at 4°C to collect the exosomes in the pellet. The size and concentration of the exosomes were determined using transmission electron microscopy and a nanoparticle tracking analyzer. Evaluate the purity of exosomes by detecting the expression levels of exosome markers such as CD81, TSG101, CD9, and calnexin using western blotting. The exosomes were labeled with a PKH67 kit (Sigma, St. Louis, MO, USA) to detect the phagocytosis of exosomes by tumor cells.