Milk Extracellular Vesicle Isolation by Differential Centrifugation

Differential centrifugation and ultracentrifugation were used for the isolation of EVs from various raw milk samples, as previously described [1 (link)]. Briefly, the defatted milk samples were subjected to differential centrifugation (210 mL starting milk volume in SW-28 rotor, Beckman Coulter, 35 mL open top tubes, Life Sciences, Boston, MA, USA) starting at 5000× g for 30 min at 4 °C. The fat layer floating on top was removed carefully. After the removal of fat globules and milk-abundant proteins such as casein and cell debris, the skimmed milk samples were subjected to successive centrifugations at 4 °C for 1 h each at 12,000× g, 35,000× g and 70,000× g. Lastly, the supernatant obtained following these spins was centrifuged at 100,000× g using a SW-28 rotor (Beckman Coulter instruments, Waverley, VIC, Australia). The pellet obtained, containing enriched milk EVs, was resuspended in sterile, filtered PBS. A final washing step was performed using ultracentrifugation at 100,000× g for 1 h at 4 °C (Beckman Coulter: TLA-55 rotor). The resulting EV pellet obtained with this method was resuspended in the appropriate volume of PBS and stored at −80°C until further analysis.