Internalization of PKH67-labeled exosomes by macrophages. (A) PKH67-labeled exosomes (20 μg) and dextran-TRITC (0.1 mg/mL) were incubated with J774 macrophages (30 minutes, 37°C) grown on glass coverslips and processed for immunofluorescence, as described. The cells were then monitored by fluorescence microscopy and transmission as indicated in the figure. PKH67-labeled exosomes were incubated with macrophages under the conditions indicated. After washing, cells were trypsinized and the fluorescence intensity was measured by flow cytometry. Tinted patterns always indicate cell autofluorescence. (B) PKH67-labeled exosomes, 10 μL (gray solid line), 30 μL (dashed line), 80 μL (dotted line), 100 μL (solid line) were incubated with J774 macrophages for 4 hours at 37°C. (C) PKH67-labeled exosomes (30 μL) were incubated with J774 macrophages for 1 hour (dashed line), 2 hours (dotted line), or 4 hours (solid line) at 37°C. (D-E) PKH67-labeled exosomes (30 μL) were incubated with peritoneal or J774 macrophages for 4 hours at 37°C with 80μM dynasore (dashed line), 5 μg/mL cytochalasin B (dotted line), or with the carrier dimethyl sulfoxide (solid line). (F) PKH67-labeled exosomes (30 μL) were incubated with J774 macrophages for 4 hours at 4°C (dashed line) or 37°C (solid line).