Mouse hepatocyte-derived extracellular vesicle proteome analysis

Mouse hepatocyte AML12 cells were plated in T175 flasks until they reached >90% confluency after which spent medium was removed, and the cells were rinsed twice with Hanks Balanced Salt Solution (ThermoFisher, Waltham, MA, United States) prior to incubating them in serum-free medium overnight, followed by replacement with fresh serum-free medium for 48 h. The supernatants were subjected to sequential centrifugation (300 × g for 10 min, 2,000 × g for 20 min, 10,000 × g for 30 min) and ultracentrifugation (100,000 × g for 70 min at 4◦C) in a Type T70i fixed-angle rotor, the pellet from which was resuspended and subjected to the same ultracentrifugation conditions again. The resulting EV pellet was dispersed in PBS, followed by protein concentration measurement by BCA assay. 5 ug of proteins were sent for mass spectrometry analysis.