EVs were isolated from plasma using differential ultracentrifugation (UC) as described previously [27 (link)] or the miRCURY Exosome Serum/Plasma Kit (Qiagen, Hilden, Germany).
In the case of UC, 500 μL plasma samples were transferred into 15 mL sterile high-speed centrifuge tubes (VWR-Avantor, Philadelphia, PA, USA), filled up with sterile 1× phosphate-buffered saline (PBS) and centrifuged twice at 10,000× g for 30 min at 4 °C in a Sorvall RC 6 Plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Supernatants were then transferred into UC tubes (Beckman Coulter, Brea, CA, USA), equilibrated with sterile 1× PBS, and spun twice at 70,000× g for 1 h at 4 °C in the Sorvall WX Ultra 100 centrifuge (Thermo Fisher Scientific). The EV enriched pellets were resuspended in 100 μL sterile PBS and stored at −80 °C until used. EV enrichment with the miRCURY Kit was performed as described [25 (link)]. Debris was cleared from 500 μL plasma samples with thrombin and subsequent centrifugation at 10,000× g for 5 min at RT. Samples were then incubated with Precipitation Buffer overnight at 4 °C and centrifuged twice (500× g, 5 min at RT). Supernatants were discarded, EV enriched pellets were resuspended in 270 μL of Resuspension Buffer and stored at −80 °C until used.
In the case of UC, 500 μL plasma samples were transferred into 15 mL sterile high-speed centrifuge tubes (VWR-Avantor, Philadelphia, PA, USA), filled up with sterile 1× phosphate-buffered saline (PBS) and centrifuged twice at 10,000× g for 30 min at 4 °C in a Sorvall RC 6 Plus centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Supernatants were then transferred into UC tubes (Beckman Coulter, Brea, CA, USA), equilibrated with sterile 1× PBS, and spun twice at 70,000× g for 1 h at 4 °C in the Sorvall WX Ultra 100 centrifuge (Thermo Fisher Scientific). The EV enriched pellets were resuspended in 100 μL sterile PBS and stored at −80 °C until used. EV enrichment with the miRCURY Kit was performed as described [25 (link)]. Debris was cleared from 500 μL plasma samples with thrombin and subsequent centrifugation at 10,000× g for 5 min at RT. Samples were then incubated with Precipitation Buffer overnight at 4 °C and centrifuged twice (500× g, 5 min at RT). Supernatants were discarded, EV enriched pellets were resuspended in 270 μL of Resuspension Buffer and stored at −80 °C until used.
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