Isolation of Extracellular Vesicles

MSC supernatants where thawed and subjected to sequential centrifugation for EV isolation. The MSC supernatants were filtered (0.2µm) and transferred into separate 50 ml falcon tubes (not mixing the different groups) prior to the centrifugation step 1 (500g, 5 min). Then, the supernatant was again transferred into a 50 ml falcon tube for centrifugation step 2 (1500g, 15 min, 4°C). Finally, supernatant was transferred into ultracentrifuge tubes (max. 38 ml per tube) for centrifugation step 3 (100,000g, 90 min, 4°C) using a Sorvall WX 80 ultracentrifuge (Beckman Coulter Inc., CA) with swinging rotor (SW32 Ti, Beckman Coulter Inc., CA). Filtered (0.22µm) dH2O was used to dilute sample to a total volume of 38ml, since the ultracentrifuge tubes need to be filled almost to the top of the tubes, to prevent disturbance of centrifugation and breaking of tubes while processing. The resulting pellet was resuspended in 1 ml phosphate buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) and stored in -20°C.

Category