EVs isolated by ultracentrifugation were further purified by iodixanol density cushion centrifugation as described by Crescitelli et al 15 . Both large and small EVs were thawed on ice and combined together.
The EVs were then mixed with 3 mL of 60% Optiprep (Sigma Aldrich) at the bottom of an Ultra-Clear centrifuge tube (Beckman Coulter), and 4 ml of 30% Optiprep and 4 ml 10% Optiprep were laid on top.
Samples were ultracentrifuged at 180,000 × g (38, (Beckman Coulter, k-factor 145) for 2 hours at 4°C. The samples were collected from the interface of the 10% and 30% iodixanol layers and stored at -80°C.
The EVs were then mixed with 3 mL of 60% Optiprep (Sigma Aldrich) at the bottom of an Ultra-Clear centrifuge tube (Beckman Coulter), and 4 ml of 30% Optiprep and 4 ml 10% Optiprep were laid on top.
Samples were ultracentrifuged at 180,000 × g (38, (Beckman Coulter, k-factor 145) for 2 hours at 4°C. The samples were collected from the interface of the 10% and 30% iodixanol layers and stored at -80°C.
