Traffic of exosomes internalized by BMDCs. (A, B) PKH67 exosomes (green) were rapidly internalized into early endosomes labeled with Texas red–transferrin (yellow indicates colocalization of green and red). (C,D) Later, PKH67 exosomes trafficked to late endosome/lysosomes labeled by Dil-LDL (red). (E) The traffic of PKH67-labeled exosomes through late endosomes/lysosomes was confirmed by colocalization of PKH67 and LAMP-1 in cytospins of BMDCs. (F) Exosomes (BALB/c) labeled with 5-nm gold (arrows) used to study the traffic of internalized exosomes within BMDCs by electron microscopy. (G) After 20 minutes, 5-nm gold exosomes (arrows) were detected in MVB expressing LAMP-1 (12 nm gold, arrowheads). No exosomes were found attached to the DC surface (asterisk). (H) Detail of an MVB expressing LAMP-1 (12-nm gold) in the limiting membrane (arrowhead) and with internalized 5-nm gold exosomes (arrows). Insert in panel H is the membrane of an internalized 5-nm gold exosome (arrow) and the membrane of the MVB (arrowhead). (I) Diagram of panel H: (1) 12-nm gold LAMP-1 on the membrane of the MVB, (2) internalized 5-nm gold exosomes, and (3) membranes of the 5-nm gold exosome (arrows) and the MVB (arrowhead). (J) Later, 5-nm gold particles (arrows, inset) were found in lysosomes stained with 12-nm gold LAMP-1 (arrowheads). (A-D) Confocal and (E) fluorescence microscopy; nuclei were stained with DAPI. (F-J) Electron microscopy; ×100 000-150 000. Bars = 100 nm. Data are representative of 4 independent experiments.