Journal: Nature Microbiology
Article Title: Phosphatidylserine-exposing extracellular vesicles in body fluids are an innate defence against apoptotic mimicry viral pathogens
doi: 10.1038/s41564-024-01637-6
Figure Lengend Snippet: a , Workflow for purification of EVs by combination of TFF and BE-SEC. See Extended Data Fig. 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 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. and Supplementary Table 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. ).
Article Snippet: The pre-concentrated sample was subsequently loaded onto two daisy-chained BE-SEC columns (bind-elute size exclusion chromatography; HiScreen Capto Core 700 column, Cytiva) connected to an ÄKTAstart chromatography system (Cytiva) with UNICORN start 1.2.0.164, which was primed (3 ml min −1 flow rate) with 500–1,000 ml sterile PBS beforehand.
Techniques: Purification, Western Blot, Protein Concentration, Staining, Concentration Assay, Derivative Assay, Flow Cytometry, Marker
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