Activity and characterization of exosomes isolated from uninfected and M bovis BCG–infected J774 cells. (A) Morphologic characterization of exosomes by transmission EM. Representative EM of a sucrose gradient purified exosome (magnification, × 100 000; scale bar = 100 nm). Images were acquired with Microtek Scan Wizard 5 (Microtek Lab, Carson, CA). (B) Surface phenotype of exosomes by flow cytometry. Sulfate/aldehyde latex beads coated with indicated exosome preparations were probed for CD86, CD81, LAMP1, LAMP2, and MHC class II, and the mean fluorescence intensity (MFI) shown (filled peaks). Open peaks represent background MFI of exosome-coated beads probed with isotype control mAb. Data are the representative of 2 separate experiments. Exosomes derived from uninfected or M bovis BCG–infected J774 cells were added to BALB/c BMMs and TNF-α (C) or RANTES (D) production was evaluated 24 hours after exosome treatment by ELISA. (E) BALB/c BMMs were treated with 10 μg of exosomes isolated from uninfected or M bovis BCG–infected J774 cells. Western blot analyses were performed on macrophage cell lysates obtained 24 hours after exosome treatment using antibodies specific for iNOS. Total p38 was used as a loading control. Results are representative of 2 separate experiments. (F) BALB/c BMMs were incubated with the exosome-containing or exosome-depleted material from the sucrose gradient. TNF-α levels were measured by ELISA 24 hours after exosome treatment. ELISA data are representative of 2 independent experiments and are expressed as means plus or minus standard deviation of duplicate wells. RC indicates resting cells; and UI, exosomes from uninfected J774 cells.