Fresh unpasteurized milk (2 liters) was collected from the Weizhu Dairy located in Nanjing, China and stored at 4 °C for 2 hours before the isolation of exosome.
Exosomes were isolated from milk using the differential centrifugation as described previously.10,29 (link) Briefly, milk was centrifuged at 3000 × g for 15 min at 4 °C to remove fat globules, cellular debris and somatic cells. The whey was collected by passing through a cheese cloth and transferred into polycarbonate tubes. In order to remove large particles, it was centrifuged for 60 min at 100 000g in Type 45Ti fixed angle rotor using Optima LE-80K Ultracentrifuge (Beckman Coulter, Brea, California). Supernatant was finally centrifuged at 135 000 × g for 90 min at 4 °C. Then the final supernatant was discarded and the exosome pellet was washed twice using the phosphate-buffered saline (PBS). The exosome pellet was re-suspended in PBS, followed by filtration through a 0.22 μm Steritop filter (Millipore, Darmstadt, Germany). The total protein concentration was determined using the bicinchoninic acid (BCA) kit (Beyotime Biotechnology, China). The concentration of the milk derived exosomes was adjusted to 6 mg ml−1 and stored at −80 °C until further use. The isolated exosomes were characterized as recommended by the International Society of Extra Cellular Vesicles (ISEV).30 (link)
Exosomes were isolated from milk using the differential centrifugation as described previously.10,29 (link) Briefly, milk was centrifuged at 3000 × g for 15 min at 4 °C to remove fat globules, cellular debris and somatic cells. The whey was collected by passing through a cheese cloth and transferred into polycarbonate tubes. In order to remove large particles, it was centrifuged for 60 min at 100 000g in Type 45Ti fixed angle rotor using Optima LE-80K Ultracentrifuge (Beckman Coulter, Brea, California). Supernatant was finally centrifuged at 135 000 × g for 90 min at 4 °C. Then the final supernatant was discarded and the exosome pellet was washed twice using the phosphate-buffered saline (PBS). The exosome pellet was re-suspended in PBS, followed by filtration through a 0.22 μm Steritop filter (Millipore, Darmstadt, Germany). The total protein concentration was determined using the bicinchoninic acid (BCA) kit (Beyotime Biotechnology, China). The concentration of the milk derived exosomes was adjusted to 6 mg ml−1 and stored at −80 °C until further use. The isolated exosomes were characterized as recommended by the International Society of Extra Cellular Vesicles (ISEV).30 (link)
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