Ti70 fixed angle rotor

EVs from cell culture supernatants were isolated by ultracentrifugation. Briefly, cells were grown in DMEM medium with exosome-depleted 5% FBS (Gibco Thermo Fisher Scientific). The media (~100 mL) was collected and pre-cleared by centrifugation at 500 × g for 10 min at 4 °C, then the supernatant was removed and discarded. After suspension in PBS, a second round of centrifugation at 2500 × g for 20 min at 4 °C to remove cell debris was performed. The supernatant was filtered through a 0.22 μm filter to remove larger microvesicles and then ultracentrifuged at 135,000 × g for 120 min (Optima XPN-100 ultracentrifuge, Beckman Coulter) using a Beckman Ti70 fixed-angle rotor. The EV pellet was washed with PBS followed by a second step of ultracentrifugation at 135,000 × g for 2 h at 4 °C, as recommended by MISEV2018 [42 (link)].

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)].