Proinflammatory activity of exosomes is dependent on the time of isolation from M bovis BCG–infected J774 cells. (A) Exosomes were isolated from the cell-conditioned media of uninfected (UI) J774 cells or at indicated times after infection with M bovis BCG and the isolated exosomes (10 or 20 μg) added to BALB/c BMMs. Cell-conditioned media collected 24 hours after exosome treatment were assayed for TNF-α production by ELISA. Data are representative of 2 independent experiments and are expressed as means plus or minus standard deviation for duplicate wells. (B) Exosomes (10 μg) isolated from the cell-conditioned media at the times indicated were analyzed for the mycobacterial components LAM and the 19-kDa lipoprotein, and the host components hsp70 and MHC class II by Western blot using antigen-specific antibodies. (C) Densitometry in arbitrary units for the bands shown in the Western blot for the 19-kDa lipoprotein and LAM. (D) J774 cells were left uninfected or were infected with M bovis BCG for 24, 48, 72, and 96 hours. Exosomes were isolated and resuspended in equal volumes of PBS and protein concentration (mg/mL) determined by Micro BCA assay. The data are representative of 2 independent experiments and expressed as means plus or minus standard deviation for triplicate wells.