Objective:To isolate and characterize macrophage-derived exosomes for constructing a stable isolation protocol for future relevant research

and to provide a reference for current isolation of exosomes. Methods:Exosomes were isolated from the culture supernatant of murine macrophages by ultracentrifugation and filtration.The size and zeta potential of exosomes were determined by a Malvern Zetasizer instrument. The morphologic features were observed with a transmission electron microscope. The content of exosomes was measured using a BCA kit. Western blot was applied to determine the expression of the marker proteins CD9 and CD63. Results:The exosomes had a size of 97.3 ± 5.73 nm and a zeta potential of-21.26 ± 1.72 according to the Malvern granulometer.They were homogeneous

saucer-like

and membrane-like microvesicles with a diameter of about 100 nm under transmission electron microscopy. Approximately 64 μg exosomes were collected from 20 mL culture medium. Western blot showed high expression of CD9 and CD63 in the exosome isolates

but no expression in the cell supernatant after isolation. Conclusion:We successfully isolated exosomes from murine macrophages by ultracentrifugation and filtration

with homogeneous morphology and high content and purity.