Isolation and Characterization of Bovine Milk-derived Small Extracellular Vesicles

Bovine milk sEVs (BEVs) were isolated from skim milk from a local grocery store by using sequential ultracentrifugation and authenticated by using Nanosight NS300 nanoparticle size analysis, scanning electron microscopy and transmission electron microscopy as previously described (Supplementary Figure 1) (32 (link)). The antibodies and their dilutions used in immunoblot analysis were the same as previously described (32 (link)). Protocol details were deposited in the EV-Track database (ID EV210338). BEVs were suspended in sterile phosphate-buffered saline (PBS) and kept at −80°C until use. For transport studies in cell monolayers, BEVs were labeled with FM 4-64 (Molecular Probes, Inc., Eugene, OR, United States) or by labeling RNA cargos by using the ExoGlow-RNA™ EV Labeling Kit (System Biosciences, Inc., Palo Alto, CA, United States) following the manufacturers’ recommendations. For transport studies in dual-chambers, BEVs were loaded with synthetic IRDye-labeled miR-34a as previously described (14 (link)).