Fig. 2.

Plasma extracellular vesicles increase after one tobacco smoking session expressed by immune and endothelial cells. Identification of red fluorescent silica beads via flow cytometry. A size range from 300 to 1000 nm was applied for size calibration (A). EV pellets were labeled by protein-specific fluorescent dye (CFSE), and by lipid-specific (FM 4-64FX) dye prior to measurements. Events within the double positive gate were identified as microvesicles (B). t-distributed stochastic neighbor embedding (t-SNE) plots of the mean fluorescence intensity of CD3, CD4, CD8, CD19, CD15, CD14, CD16 and CD56 surface antigens in a total of 50,000 double positive events (C). Floating bar charts showing the concentration of protein before and after the tobacco smoking intervention, measured via Bradford assay (D). Representative gating strategy of the MACSPlex exosome assay, showing the bead size selection (right) and the 37 different dye-labeled capture beads against 37 different EV surface antigens based on FITC and PE fluorescence (left) (E). Heatmap of CD9⁻CD63⁻CD81 normalized MFI in all individual samples, showing 19 differentially expressed surface markers in our cohort both before and after the intervention (F). Violin plots showing the mean MFI of the three main EV markers CD9, CD63 and CD81 in each cohort (far left) and the signal intensity of CD105, CD49e and CD31 surface antigens after detection of CD9, CD63 and CD81 (G). Detection of EV (CD63), endothelial (CD105), cell lysate (GRP94) and loading control (β-actin) markers verified by western blotting before (pre), and 60 min after smoking (post) (H). Statistical evaluations were performed using Wilconxon matched-pairs rank test. P values: * for p ≤ 0.05; ** for p ≤ 0.001; *** for p ≤ 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)