Depletion of EVs from Fetal Bovine Serum

The depletion of EVs in FBS was carried out using a methodology proposed earlier [33 (link)]. In brief, FBS was ultrafiltered using Amicon Ultra-15 centrifugal filter devices (100 kDa cutoff, MerkMillipore, Darmstadt, Germany) for 30 min at 5000× g. The filtrate was collected and measured for particle concentration using NTA. For this study, the vesicle-depleted conditioned medium was used to isolate and purify EVs by SEC, and the EV preparations met the optimal requirement as per the guidelines prescribed by the International Society for Extracellular Vesicles (ISEV) [34 (link)].