Sucrose gradient fractionation of EVs

Purified EVs were subjected to continuous sucrose gradient fractionation using established procedures ^31,63. Briefly, twelve stock solutions with decreasing sucrose concentration were layered on top of purified EVs (800 μg, in 100 μl PBS) in a total volume of 12 ml, followed by ultracentrifugation at 100000 x g for 16h at 4°C with a Beckman ultracentrifuge and a SW40Ti rotor. Six fractions of 2 ml each were then collected and transferred to new tubes; the density of each sucrose fraction was measured using a refractometer. Each fraction was washed with 10 ml of PBS and ultracentrifuged at 110000 x g for 70 min at 4°C as above. EVs were resuspended in 100 μl of PBS prior to subsequent analyses.