Exosomes, not apoptotic vesicles, are responsible for the proinflammatory activity. (A) J774 cells were infected with M bovis BCG for 4 hours. As controls, macrophages were left uninfected or were starved of serum to induce apoptosis. Macrophages were treated with annexin V (green) and propidium iodide (red), fixed, and analyzed by confocal microscopy. Nuclei were stained using the bisalkylamino-anthraquinone fluorphore Draq5. Fluorescent images are representative of 2 independent experiments (scale bar = 10 μm). Images were acquired with a Nikon Diaphot 200 (Nikon, Tokyo, Japan), equipped with a 60× oil plan Apo160/0.17 NA lens with BioRad LaserSharp 2000 software (BioRad, Hercules, CA). (B) Exosomes were prepared from J774 cells infected with M bovis BCG treated with 50 μM of the caspase-3 inhibitor Ac-DEVD-CHO, or left untreated. As a control, apoptotic vesicles were also purified from serum-deprived J774 cells. Exosomes or apoptotic vesicles were added to separate, uninfected J774 cells; 24 hours after treatment, cell-conditioned media were assayed for TNF-α by ELISA. Data are representative of 2 independent experiments and are expressed as means plus or minus standard deviation for duplicate wells. (C) Cell lysates obtained 24 hours after treatment with exosomes or apoptotic vesicles were analyzed for iNOS expression by Western blot. Total p38 was used as a loading control. The results are representative of 2 separate experiments. (D) Sulfate/aldehyde latex beads coated with exosomes or apoptotic vesicles were probed by flow cytometry for annexin V binding or FcRII/III, and the percentage of beads positive for the markers is shown. Results are representative of 2 independent experiments and are exposed as means plus or minus standard deviation for duplicate wells. UI indicates uninfected J774 cells; BCG, J774 cells infected with M bovis BCG.