Isolation and Characterization of Urinary Extracellular Vesicles

EVs were isolated from 10 mL’s of urine from diabetic db/db mice collected during the active cycle (AM collection) or inactive cycle (PM collection) while the mice were in metabolic cages. The urine samples were centrifuged at 1000× g for 15 min at 4 °C before being filtered through a 0.2 μm rapid-flow Nalgene filter (Thermo Fisher Scientific). The supernatant was then subject to ultracentrifugation at 52,000 rpm for 90 min at 4 °C using a ti-70 fixed-angle rotor (Beckman Coulter, Inc., Brea, CA, USA). The resulting EV pellet was resuspended in 200 μL of ultra-pure 1×PBS (ThermoFisher) and then aliquoted and stored at −80 °C. To measure EV size and concentration, nanoparticle tracking analysis was performed using a NanoSight NS300 (Malvern Panalytical, Malvern, UK) machine equipped with NTA 3.4 Build 3.44 Software (Malvern) as previously described by our group [43 (link)].