Fig. 1

Characterization of size-based subpopulations of EV released from TNFα/IFNγ treated (tEV) and untreated hCMEC/D3 endothelial cells (uEV). (A) EV size distribution profile (nm) and concentration (particles/ml) of both uEV (black) and tEV (red) fractionated based on their size at low (2,000×g = EV-2 K and 10,000 ×g = EV-10 K) and ultra-centrifugal force (100,000 ×g = EV-100 K) using NTA. Histograms represent the mean ± SD of nine independent biological experiments (n = 9). (B) The percentage of small EV (0- 150 nm), large EV (150–300 nm) and very large EV (> 300 nm) in the 2 K, 10 and 100 K fractions. Data were obtained from nine independent biological experiments (n = 9). (C–D) Data represent (C) the particle number per milliliter of uEV (black) and tEV (red) of size based EV subpopulations (n = 6). (D) Total amount of protein per particle uEV (black) and tEV (red) size based EV subpopulations (n = 3). Data of NTA analysis are presented as means ± SEM (n = independent biological experiments, 1 symbol per batch) and one-way ANOVA Tukey’s multiple comparison was used to determine significance between multiple groups: ns, no significance, *p < 0.05 **P < 0.01, ****P < 0.0001. (E) TEM images of uEV and tEV in different recovered sized based fractions (2 K, 10 K, and 100 K) (Scale bar = 200 nm). (F) Representative western blots of CD9 (24 kDa), CD63 (30–70 kDa) as classical EV membrane-bound markers, ICAM-1 (85–110 kDa) as inflammation-associated marker, Annexin II (38 kDa) as a cytosolic marker and a proapoptotic -associated protein (BAX ~ 25 kDa) as a negative control in EV lysate side-by-side with the lysate of producing untreated (CTRL) and inflamed (TNFα/IFNγ) cells. Five micrograms of EV proteins were loaded on the gels. The same trends were detected in western blot data of at least three independent samples (n = 3)