Path of internalization of exosomes in SKOV3 cells. (A) Exos-CFSE (20 μg protein; green) were incubated with SKOV3 cells at 37 or 4°C and uptake was monitored by flow cytometry analysis of cell fluorescence intensity. Solid and dashed lines represent Exos-CFSE uptake at 37 and 4°C, respectively. (B) Colocalization of Exos-CFSE (20 μg protein; green) with EEA1 (red), LAMP1 (red) and caveolin-1 (red). Secondary antibody was donkey anti-mouse IgG AlexaFluor 594. Colocalization is indicated in yellow. Images in the left bottom represent 4 × magnifications of selected areas. Scale bars = 10 μm. (C) Effect of chlorpromazine, cytochalasin D, EIPA and methyl-beta-cyclodextrin (30 min pre-incubation) on Exos-CFSE uptake (4, 4, 4 and 2 h, respectively) monitored by flow cytometry analysis (dashed lines). Controls consist of SKOV3 cells with no treatment for chlorpromazine and methyl-beta-cyclodextrin, or treated with DMSO for cytochalasin D and EIPA (solid lines). Unlabelled SKOV3 cells (grey) were used as control for cell autofluorescence. The results shown are representative of three independent experiments.