Macrophage-derived Extracellular Vesicles Isolation

Macrophage FTV were isolated using procedures from previous reports with some modifications [19 (link)]. Briefly, nonpolarized macrophages, M1, M2 differentiated macrophages and HG treated macrophages were initially washed by serum-free cell culture medium, followed by treatment with trypsin. Subsequently, a low-speed centrifugation at 1000 × g for 10 min, then at 2000 x g for 20 min to remove cells and cellular debris. The collected cell culture medium was then mixed with an extraction buffer (Lysosome Isolation Kit, Sigma-Aldrich, USA), These crude FTV fraction were subjected to 150 000× g for 4 h ultracentrifugation at a multi-step 2%, 5%, 8%, 10%, 12%, 15%, 19%, 25%, 30% Optiprep density gradient medium solutions. The purified FTV fraction were then carefully collected and named M0-ftv, M1-ftv, M2-ftv, HG-ftv respectively. The prepared samples are subjected to further analysis or stored at -80 °C for subsequent processing. FTV size distribution and quantification of vesicles was analyzed by Nanoparticle tracking analysis (NTA) .