Extracellular Vesicle Isolation and Characterization from Transfected 293T Cells

Human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) were grown in DMEM (Gibco) plus 10% FCS (Gibco). Transfection assays were performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific)-based method. 293T cells were transfected with vectors expressing either N or Nef^mut/N, and supernatants harvested from 48 to 72 h after transfection. EVs were recovered through differential centrifugations by centrifuging supernatants at 500 × g for 10 min, and then at 10,000 × g for 30 min. Supernatants were harvested, filtered with 0.22 μm pore size filters, and ultracentrifuged at 70,000 × g for l h. Pelleted vesicles were resuspended in 1×PBS, and ultracentrifuged again at 70,000 × g for 1 h. Afterward, pellets containing EVs were resuspended in 1:100 of the initial volume.
Western blot analyses of both cell lysates and EVs were carried out as reported^{11 (link)}, and filters were revealed using 1:2000 diluted anti-flag M2 mAb from Merck (cat. H1029), 1:500-diluted anti-β-actin mAb (cat. 122625, Cell Signaling), 1:500 diluted anti-Alix Abs (PA5-52873 Invitrogen). Filters were analyzed by a Chemi-Doc apparatus (Bio-Rad) and relevant signals quantified by Image Lab software version 6.1.