Fig 1: EV characteristics of mortality cohort. (A) EV morphology and size were visualized using electron microscopy (scale bar = 200 nm). (B) Plasma EVs from alive (n = 8) and individuals who had died (n = 8) within 5 years of sample collection were lysed and the EV markers CD81 and ALIX using quantified using ELISA assays. Individual data points are shown, and the bar represents the mean. There were no significant differences in CD81 and ALIX EV levels between alive and deceased individuals using Student’s t-test. (C) EV markers in plasma EVs were assessed using an Exo-Check™ Exosome Antibody Array. (D,E) Plasma EVs were isolated from participants who were alive or died within five years of sample donation (Table 1), and EV size distribution and concentration were analyzed by Nanoparticle Tracking Analysis. (D) Size distribution was averaged for each group (n = 75 for alive and n = 74 for died groups). EV concentration is shown in the plots in (E).
Fig 2: Fluorescent activated cell sorting (FACS) enrichment of plasma NDEVs, ADEVs, and MDEVs from LATE‐NC subjects. Representative FACS plot for non‐EV, negative control (red) and BAE—FITC complexes generated from EVs (green) isolated a LATE‐NC subject and enriched against anti‐human CD171 biotin (L1‐CAM), anti‐GLAST, and anti‐TMEM119 antibody. (A) Representative plot of size/concentration determined by nanoparticle tracking analysis (NTA) for extracted plasma EVs from a LATE‐NC subject (B). Plasma concentrations of EV marker, CD81 as measured by hu‐specific ELISA. CD81 was not statistically different between the three groups (C).
Fig 3: TNF-α does not affect production, expression of surface markers, size or morphology of endothelial-derived SEVs. (A) Immunofluorescence showing co-localization of Rab7 and CD63 in endothelial cells treated with or without TNF-α (n = 3) Scale bar: 20 μm. (B) Size distribution (n = 7), (C) concentration (n = 7), (D) modal size (n = 7) determined by Nanoparticle Tracking Analysis. (E) CD81 expression determined by ELISA (n = 4), and (F) morphology of endothelial small extracellular vesicles treated with (aSEVs) or without (cSEVs) TNF-α assessed by electronic microscopy. Scale bar: 200 nm. Mean ± SD.
Fig 4: SDS-PAGE analysis: (a) Compared with the standard liquid, the molecular mass of CD63 is 50 kDa, while that of CD81 falls within the range of 20–25 kDa. (b) Electrophoresis results of A549 EV proteins at different dilution factors. (c) Electrophoresis results of MG63 EV proteins at different dilution factors.
Fig 5: The ratio of CD63 to CD81 remains constant for EVs of the same type under various dilution factors, while variations are observed among different EV populations.
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