NK3.3 and NK-92 cells were cultured overnight with IL-2 and then treated with phorbol myristate acetate (PMA) and ionomycin for 5 h to enhance secretion of EVs into the media. HEK293 cells were grown to 90% confluency over 48 h. Media was harvested and centrifuged at 300 × g for 10 min to pellet cells. A commercial polyethylene glycol polymer for precipitation of EVs was added to the supernatants for a minimum of 12 h at 4°C (ExoQuick-TC, System Bioscience (SBI)) and were centrifuged at 3000 × g at 4°C for 10 min per a modified ExoQuick-TC protocol. EV-depleted supernatants were discarded, and EV pellets were resuspended in PBS. Protein concentrations were assessed via BCA protein assay kit (ThermoFisher Scientific).
NK3.3-derived vesicle fractions were isolated by differential ultracentrifugation (Federici et al., 2020 (link)). Briefly, NK supernatants were centrifuged at 300 × g for 10 min, followed by centrifugation at 2,000 × g for 20 min and then 10,000 × g for 30 min at 4°C. This pellet was considered to be the microvesicle (MV) fraction. Additional ultracentrifugation at 100,000 × g for 90 min at 4°C was performed to isolate the exosome fraction. All pellets were resuspended in PBS and protein quantitated by BCA.
NK3.3-derived vesicle fractions were isolated by differential ultracentrifugation (Federici et al., 2020 (link)). Briefly, NK supernatants were centrifuged at 300 × g for 10 min, followed by centrifugation at 2,000 × g for 20 min and then 10,000 × g for 30 min at 4°C. This pellet was considered to be the microvesicle (MV) fraction. Additional ultracentrifugation at 100,000 × g for 90 min at 4°C was performed to isolate the exosome fraction. All pellets were resuspended in PBS and protein quantitated by BCA.
Free full text: Click here
