Isolation and Characterization of Exosomes from Tamoxifen-Induced Lung ECs

ECs isolated from tamoxifen-induced p16^iΔEC or p16^fl/fl mouse lungs were plated (1 × 10⁶ cells per 6 cm dish), cultured with media containing exosome free FBS, and conditioned media was collected after 24 h. Exosomes were isolated using Total Exosome Isolation Reagent from Cell Culture Media (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol (41 (link), 72 (link)). The exosome pellet was resuspended in 25 μl of 0.2 μm filtered PBS. Isolated exosomes were confirmed with exosome marker proteins (CD63, flotillin-1) using immunoblotting (IB). For transmission electron microscopy (TEM) to analyze the ultrastructure of the exosome, resuspended exosomes were adsorbed onto freshly ionized, 400 mesh formvar/carbon grids, washed once with distilled water, and negatively stained with 2% aqueous Uranyl acetate. Exosome preparations were viewed in a Hitachi H600 transmission electron microscope and images were recorded with a Hamamatsu ccd camera using AMT image capture software. Size and concentration distributions of exosomes were determined using nanoparticle tracking analysis (NTA; NanoSight LM10 system, Malvern instruments, Malvern, UK) (41 (link), 72 (link)).