“Eat me” signal mediates exosome uptake by monocytes/macrophages. (A) Exosomes were collected from culture supernatant of Akata- and B95-8-LCLs by ultracentrifugation and further fractionated by iodixanol gradient centrifugation. CD63 expression in each exosome was detected by western blotting under nonreducing conditions. (B) PBMCs were treated with 2.5 mg/mL Akata and B95-8 exosomes for 48 hours. Flow cytometric analysis revealed strong incorporation of exosomes into monocytes, but not into lymphocytes. (C) Each dose of recombinant MFG-E8 and annexin V was added to the PBMCs and cultured for 4 days. Incorporation of PKH-labeled exosomes by monocytes was detected as described in Figure 4B. The inhibition of excess of MFG-E8 or annexin V on exosome uptake was repeated 5 times, using PBMCs derived from 2 individuals.