Figure 6.

Characterization of human omental tissue-derived exosomes. (A) Exosomes were isolated from the CM of human omental tissue explants and analyzed by cryogenic transmission electron microscopy (Cryo-TEM); Scale bar = 100 nm. (B) Measurement of omental tissue exosomes diameter by nanoparticle tracking analysis system; (C) Western blot analysis of the exosome markers CD81 and CD63. Representatives of three independent experiments are shown; (D) PKH-67-labeled omental tissue exosomes were incubated with AGS gastric cancer cells, reaction was stopped at different time points (1, 3, 5, and 7 h) and cells were analyzed by confocal microscopy (upper panel). The cell's nucleus was stained with Dapi. PKH-67 labeled omental tissue exosomes were incubated for the indicated time points (30 min, 1, 2, 3, 4, 5, and 6 h) with AGS gastric cancer cells. Internalization was measured by flow cytometry (lower panel). Negative control-AGS cells with no addition of labeled exosomes. The data are presented as mean ±SD of four independent experiments; (E) AGS cells were pre-treated with 80 μM Dynasore or 10 ng/mL Heparin for 30 min or with 2 μM Simvastatin for 16 h. PKH-67 labeled omental tissue exosomes were incubated with untreated (positive control) or treated cells for 3 h and uptake was measured by flow cytometry analysis (right panel). The data are presented as the mean percent of cells that uptake labeled exosomes ± SD of three independent experiments; PKH-67 labeled omental tissue exosomes were incubated with untreated (positive control) or treated cells (Dynasore, Heparin or Simvastatin) and internalization was measured by confocal microscopy (left panel). Scale bar is 10 μm.