fig2

Figure 2. Characterization of BAL EVs isolated by SEC and concentrated by various methods. Particle count as determined by Nanosight (A) and total protein by BCA (B) in each concentration method. Concentrating SEC fractions 1-6 using a 30 kDa Amicon centrifuge filter yielded equally high EV particles compared to concentration using 10 kDa centrifuge filter with lower free protein contamination. n = 1 subject run three times to demonstrate technique reproducibility. Error bars represent SEM as determined by NTA 3.3 software from Malvern Paranalytical; (C) Western Blot of Tetraspannins CD9, CD63, CD81, and Syn-1 of EVs isolated by SEC and concentrated by either Amicon centrifuge filters, EE kit, or ultracentrifugation. BAL: Bronchoalveolar lavage; EVs: extracellular vesicles; SEC: size exclusion chromatography; BCA: bicinchoninic acid; kDa: kilodalton; SEM: standard error of the mean; NTA: nanoparticle tracking analysis.