Fig. 3.

sEVs when uptaken by primary microglia follow the endocytic pathway and are colocalized with Rab5 (early endosomes-EE) and Lamp1 (late endosomes-LE/Lysosomes). Microglia cells were incubated with Dil-labeled sEVs (red), for 6 h and 24 h. Colocalization of sEVs with Rab5 and Lamp1 was monitored at 6 h and 24 h post-treatment. Cells were fixed and immunolabeled for Rab5 or Lamp1 (green), α-Tubulin (α-Tub) (gray), and DAPI (blue), detecting cell nuclei. Representative Imaris images depict colocalization between internalized sEVs and the endocytic markers, Rab5 (a–c) and Lamp1 (d–f), after 6 h and 24 h of incubation. The colocalization channel and surface (yellow) were built using the Imaris imaging software. Scale bar 5 μm. Graphs show colocalization between sEVs and Rab5/Lamp1 (Manders’ colocalization coefficient) after 6 h and 24 h (b and e) of treatment as well as the mean volume of puncta colocalized with Rab5 (c) and Lamp1 (f) at the different time points. Data are presented as the mean ± SEM of minimum three independent cell preparations, with more than 80 cells measured; Student's t test was used, and statistical significance was set as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001