Activity and characterization of exosomes isolated from uninfected, M bovis BCG–infected and M tuberculosis H37Rv–infected THP-1 cells. (A) Morphologic characterization of exosomes by transmission EM. Representative EM of a sucrose-gradient–purified exosome (magnification, × 80 000; scale bar = 100 nm). Images were acquired with BioRad LaserSharp 2000 (BioRad). (B) Surface phenotype of exosomes by flow cytometry. Sulfate/aldehyde latex beads coated with indicated exosome preparations were probed for the indicated proteins. The MFI is shown. Light-penciled peaks represent background MFI of exosome-coated beads probed with isotype control mAb. The data are representative of 2 separate experiments. (C) Exosomes (10 μg) isolated from the cell-conditioned media of uninfected or THP-1 cells infected for 72 hours with M. bovis BCG were analyzed by Western blot for the indicated host proteins as well as the mycobacteria 19-kDa lipoprotein and LAM. (D) Exosomes isolated from uninfected, M bovis BCG–, or M tuberculosis H37Rv–infected THP-1 cells were added to separate uninfected THP-1 cells; 24 hours after treatment, cell-conditioned media were assayed for TNF-α by ELISA. Data are representative of 3 independent experiments and are expressed as means plus or minus standard deviation for duplicate wells. (E) Cell lysates obtained 24 hours after exosome treatment were analyzed for iNOS expression by Western blot. Total p38 was used as a loading control. Results are representative of 2 independent experiments