Fig. 3: EVs from body fluids expose PS at varying levels.
a, Workflow for purification of EVs by combination of TFF and BE-SEC. See Extended Data Fig. 3a,b for elution profiles and size distributions. b, Western blot analysis for verification of EV-associated proteins enrichment by TFF/BE-SEC in protein-concentration-adjusted samples. See Supplementary Information for uncut membranes, markers and total protein staining. Representative blots of n = 3 repetitions with similar results for blood, saliva and semen, and n = 1 for urine and milk. c, Major phospholipid distribution in purified EVs analysed by shotgun lipidomics. Shown are means of two biological replicates with averaged technical duplicates (saliva EVs) or two to three technical replicates of an individual EV preparation (other sources), each from body fluids pooled from at least 35 (semen), 9 (saliva), 6 (urine), an individual (milk) or 50 donors (blood). See Extended Data Fig. 3c and Supplementary Table 3 for extended lipidomics data. d–f, Concentration of PS (d), PE (e) and PC (f) according to lipidomics normalized to NTA-based particle concentration per source. One-way ANOVA (95% confidence interval) with Bonferroni’s post-test compares semen and blood-derived EVs. g, PS + PE to PC ratio based on d–f. All in d–g based on lipidomics data from c showing means ± s.d. h, Surface exposure of PS analysed by bead-assisted flow cytometry for EV subpopulations also exposing the indicated marker proteins. Shown are means of averaged technical duplicates for n = 5 (saliva), n = 3 (semen, blood) or n = 1 (urine, milk) individual EV preparations from pooled sources ± s.d. i, Proportion of PS-exposing TSPN-positive (CD9/CD63/CD81 and PS double positive) vesicles in semen EV preparations detected by nanoscale flow cytometry. n = 5 biological replicates (individual EV purifications from distinct pooled sources), means ± s.d. (Extended Data Fig. 3e).
