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Fluorescent lipid probes were used to track lipid trafficking between parent RBL cells and exosomes. We have checked the intracellular labeling of exosomes ("in vivo labeling") from parent cell incubated with either Bodipy-Cer, Bodipy-PC, or NBD-PC. Bodipy-PC labeled equally cells and exosomes, whereas Bodipy-Cer, a Golgi marker, was enriched in exosomes. Golgi membranes participated effectively in exosome biogenesis since cell incubation with brefeldin A leads to a modified phospholipid/protein ratio in exosomes. At the opposite, NBD-PC, a plasma membrane marker weakly labeled exosome membranes. Sorting of subpopulations indicated that the MHC-II containing exosomes were enriched in Bodipy-PC, whereas tetraspanin(CD 63 or CD81)-containing exosomes are essentially labeled with Bodipy-Cer and Bodipy-PC. These results indicated that RBL released two main subpopulations of exosomes that can be discriminated by their protein and lipid contents. When the bulk of exosomes was labeled after their purification ("in vitro labeling") with either of the above-mentioned lipid probes, the Bodipy-Cer was the only one to incorporate noticeably in all the subpopulations, indicating that the previous results obtained during "in vivo labeling" monitored real intracellular lipid trafficking between organelles and exosomes. Bodipy-Cer was further used as a tool to measure the respective amounts of each subpopulations. CD63, MHC II, and CD81-containing exosomes accounted for 47%, 32%, and 21%, respectively, of total exosomes.