Extracellular Vesicle Isolation Protocol

EVs were isolated from HEK-293T cell complete cell culture media as described previously [25 (link)]. Before isolation, HEK-293T cells were cultured in complete DMEM media supplemented with FBS depleted of animal EVs by ultrafiltration using Amicon Ultra-15 100 kDa filter devices (Merck Millipore, Darmstadt, Germany), as shown in [26 (link)]. In brief, conditioned media were centrifuged at 2000× g for 10 min and then at 10,000× g for 10 min to remove cell debris and large EVs followed by anion-exchange chromatography using MacroPrep DEAE Media anion-exchange resin (BioRad, Hercules, CA, USA). The column was washed with 100% buffer A (50 mM HEPES, 100 mM NaCl) followed by stepwise washing with 95% buffer A/5% buffer B (50 mM HEPES, 2 M NaCl)—5 column volumes (CV), 90% buffer A/10% buffer B—10 CV. The fraction containing EVs was eluted with 60% buffer A/40% buffer B and the column was then washed with 100% buffer B. The eluate was concentrated using Amicon Ultra-15 (100 kDa) filter devices followed by 3 washes with PBS and 1 more concentration step.