Characterization of CD3⁺–immune-selected CD19.CAR⁺EVs. NTA (A) and AFM analysis (B). (C) Flow cytometry evaluation of CD63 and flotillin-1 expression. CD63 and flotillin-1 expression (red histograms) were analyzed using the related FMOs as controls (blue histograms). MFI ratio values were calculated by dividing the MFI of the positive sample and that of the related FMO control. Data are representative of 3 separate experiments. (D) Flow cytometry killing assays were performed using the concentration of 0.015 μg of protein from CD3-immunoselected EVs per target cell to treat both Raji and SUP-B15 cell lines and by analyzing their cytolytic activity by measuring the 7-AAD staining of target cells after 24 hours of treatment.