EV Isolation and Characterization Protocol

EV isolation and characterization were previously described [14 (link),15 (link)]. Briefly, NK3.3 cells were cultured overnight in RPMI-1640 media with 3% EV-depleted FBS and 200 U IL-2/mL and then treated with phorbol myristate acetate and ionomycin for 5 h to augment EV release. HEK293 cells were grown to 90% confluency over 48 h in DMEM and 3% EV-depleted FBS. NK3.3 cell suspensions and HEK293 culture media were centrifuged at 300× g for 10 min to pellet cells and debris. Supernatants were removed, centrifuged again at 2000× g for 10 min to remove smaller debris, and passed through a 0.22 μm filter. A commercial polyethylene glycol polymer for EV precipitation was added to the supernatants for a minimum of 12 h at 4 °C (ExoQuick-TC, System Biosciences, Palo Alto, CA, USA). After incubation, supernatants were centrifuged at 3000× g at 4 °C for 10 min per a modified ExoQuick-TC protocol. EV pellets were resuspended in phosphate-buffered saline (PBS). Protein concentrations were assessed using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA); particle numbers and sizes were determined by nanoparticle tracking analysis (NTA) (NanoSight NS3000, Malvern Panalytical, Malvern, UK). There was minimal batch-to-batch variation in EV preparations made by PEG precipitation. For both HEK293 and NK3.3 EVs, 1 μg of protein consisted of 1–2 × 10¹¹ EV particles.