Urine Extracellular Vesicle Isolation

PEG precipitation and subsequent UC was performed as described previously [31 (link)], with some minor modifications. Briefly, pre-processed urine samples were centrifuged for 45 min at 10,000× g and 4 °C. Following addition of 1 mL 50 w/v % PEG-6000 (Sigma-Aldrich, Steinheim, Germany) and 750 µL of 0.9% NaCl (Fresenius Kabi, Bad Homburg, Germany) to 8.25 mL supernatant (final PEG concentration: 10%), uEVs were precipitated at 4 °C overnight. Precipitates were pelleted by centrifugation for 30 min at 1500× g and 4 °C. Pellets were resuspended in 0.9% NaCl and centrifuged at 110,000× g for 2 h at 4 °C in an XPN-80 ultracentrifuge equipped with a Type 50.4 Ti rotor (Beckman Coulter, Krefeld, Germany; k-factor: 93). UC pellets were resuspended in 1 mL of 0.9% NaCl and stored at −80 °C until further analysis.