Fusion/hemifusion of exosomes with BMDCs. (A) Structure of R18-labeled exosomes purified by gel filtration (×100 000). Bar = 100 nm. (B) Spectrofluorimetric analysis after addition of (unlabeled) BMDCs to R18-exosomes. When R18-exosomes were incubated alone (control) fluorescence increased only after their disruption with Triton X-100 (T X100). (C-F) Fusion assays of R18-exosomes with BMDCs at different conditions. (B-F) Results are expressed as percentages of maximal fluorescence de-quenching [FD (%)]. Spectrofluorimetric assays were done exposing 10⁶ BMDCs to 10 μg of R18-exosomes in a final volume of 2 mL of 2 g/L glucose Ca/Mg HBSS. (G) Differential interference contrast (top) and fluorescence (bottom) images during fusion/hemifusion of R18-exosomes with BMDCs. Arrow indicates the fusion/hemifusion of one R18-exosome(s) with the BMDC on the right, detected when the area of diffusion of de-quenched R18 on the DC surface reached the limit of resolution of the objective. Images were pseudo-colored to indicate the intensity of light. Arrowhead points to another fusion of an R18-exosome(s) with a neighboring BMDC. The chart shows the quantitative analysis of the cells from the images (×400). For fluorescence time-lapse microscopy, 150 000 BMDCs were incubated with 2.5 μg of R18-exosomes in a final volume of 500 μL. Results (B-G) represent ≥ 3 experiments per condition.