Ultracentrifugation-based Extracellular Vesicle Isolation

We used ultracentrifugation without a sucrose-gradient centrifugation step to isolate EVs from MSCs. EVs were isolated from approximately 100 mL of EM after 48 h of incubation of ~2 × 10⁷ MSCs using several ultracentrifugation steps, as described [16 (link)]. EV protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) to ensure equal amounts of protein samples for experiments. EVs were suspended in RIPA buffer (1% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate in Tris-buffered saline (TBS), Sigma-Aldrich) for Western blotting or in PBS for characterization and functional analysis. NTA and electron microscopy were performed as described [68 (link)]. Pre-embedding immunogold staining was performed by incubating sections in the primary antibody anti-CD63 (Abcam, TS63, Cambridge, UK) and then in colloidal gold-conjugated secondary antibody (anti-IgG mouse, Abcam).