Fig. 5

Confocal micrographs of internalization of MV derived from unstimulated HBMEC by sex-matched unstimulated HBMEC. MV generated from female donor cells were first labeled with PKH67 (green), then rinsed and applied to naïve female recipient cells for various time intervals; non-MV-treated cells served as controls. At each time point, 30 min, 90 min, or 20 h, paired sets of control and MV-exposed cells were rinsed to remove non-internalized MV and labeled with markers for (1) early endosome antigen-1 (EEA-1) conjugated with IgG/AlexaFluor 647 IgG (cyan), (2) lysosomes [LysoTracker™ (red)], and (3) nuclei [DAPI (blue)] as described in the “Materials and methods” section. a Merged maximal intensity projections of cells not treated (0 min) or treated with PKH67-labeled MV for 30 min, 90 min, or 20 h. Sparse PKH67-positive punctate structures were present in the cytoplasm 30 min after MV application and were increased in number after 90 min, most of which were co-labeled for lysosomes (yellow). MV labeling was reduced at 20 h. Co-localization of MV/early endosome labels was absent at all time points examined. b The image of the 90-min MV-treated cells (above) was used to substantiate accurate assessment of localization of each fluorescent marker. Images obtained by sequential scanning of individual channels for PKH67, EEA-1, and LTR are shown, together with the resulting merged image which also includes the DAPI channel. The region bounded by white box in the merged image from 90 min was enlarged (c), and a representative fluorescence intensity profile along the dashed line encompassing labeled cytoplasmic structures showed overlapping signals for PKH67 with LTR, but not with early endosomes (d). White bars, 5 μm; except in c, 1 μm