Sterile conical tubes

Void urine samples were collected from healthy Caucasian adult male and female volunteers following obtained oral and written informed consent. The study was approved by the ethics committee of the Medical Faculty of the University of Duisburg-Essen (no. 18-8494-BU). Random spot urine was collected in sterile, leak-proof plastic containers (Sarstedt, Nümbrecht, Germany) and the absence of leukocytes, nitrite-producing bacteria, blood, or protein was confirmed by a urinary dipstick test (Multistix 10 SG, Siemens Healthineers, Erlangen, Germany). After collection, the samples were immediately processed. They were first centrifuged at 500× g for 10 min (4 °C) and 3000× g for 20 min (4 °C) to remove cells, cellular debris, and larger vesicles. Supernatants were stored at −80 °C in 10 mL aliquots in sterile conical tubes (Falcon, Corning, NY, USA) until further processing unless otherwise indicated. Details of the centrifugation procedures are provided in Supplementary Table S4.
Urine samples considered for direct IFCM analyses without previous sEV enrichment were pre-cleared by filtration before antibody staining using 0.22-µm polyethersulfone syringe filters (Minisart, Sartorius, Göttingen, Germany).

Hydrolysate was centrifugation (27,000×g, 30 min), the supernatant was vacuum filtered through a Buchner funnel with glass filter paper (Watman, GF/C) and then through a SteriCap Bottletop Filter Unit (0.22 µm PES membrane, 40 cm² filtration area, 5–10 L capacity) (Merck-Millipore) in an aseptic laminar flow hood. The final sterile hydrolysate was aliquoted into 50 mL sterile conical tubes (Falcon) and stored at − 20 °C.