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Fig. 5 Internalization of pEVs by HMC3 microglial cells after their passage through bEnd.3 endothelial cells. (a) HMC3 cells were cultured at the bottom without or (b) with bEnd.3 cells seeded on a Transwell for 10 days, then 1 1010 pEVs/mL were added at the luminal side for 24 h. (c) Internalization of PKH-pEVs (in red, indicated by arrows) by HMC3 cells (nuclei in blue, Iba1 in green) at 4°C and 37.5°C after 1-h and (d) 24 h incubation with or without bEnd.3 cells on the Transwell device. (e) Internalization of PKH-pEVs (in red) by microglial cells (nucleus in blue, Iba1 in green) confirmed by a z-axis plan (40×). (f) Percentages of HMC3 cells internalizing PKH-pEVs were quantified by flow cytometry and normalized according to the incubation of microglial cells with an insert and pEVs CTL in the luminal compartment. Results were compared by ordinary one-way ANOVA and Tukey's multiple comparisons test. (g) Percentages of HMC3 cells internalizing PKH-pEVs were quantified by flow cytometry. (h) Levels of IL-6 on pEV samples by ELISA and comparison by ordinary one-way ANOVA, Tukey's multiple comparisons test. (i) Quantification of IL-6 released by HMC3 cells in the media following 24 h of treatment with either PBS, LPS, Aβ or pEVs. Comparison by ordinary one-way ANOVA, Tukey's multiple comparisons test. (j) Levels of IL-6 released by HMC3 cells in the presence of bEnd.3 cells and were normalized according to the amount transported by pEVs mAD. IL-6 levels were quantified into both compartments of the Transwell device and compared by ordinary one-way ANOVA, Tukey's multiple comparisons test. Prior statistical analysis, the normality of each data set was evaluated. Data are expressed in mean ± SEM with *p ≤ 0.5; **p ≤ 0.05; ****p ≤ 0.0005 with n ≥ 6 per group.