FIGURE 3.

SEVEN for characterization of SEC‐enriched EVs from pooled human plasma. (a) Left, Tetraspanin‐enriched EVs were detected using CF568‐maleimide (labels available cysteine residues on the membrane, yellow) and AF647‐labelled anti‐TSPAN Abs (labels membrane tetraspanins, red). Right, 2D distribution of relative centroid shifts for two‐colour EVs (centroid of the yellow channel is assigned  0,0 position). Full width at half maximum values for Gaussian fittings of EV counts are shown; n = 3. (b) Number of detected TSPAN‐enriched EVs per ROI. The x‐axis represents EV concentrations normalized to the highest applied EV concentration (undiluted SEC‐enriched EVs). Mean ± SEM; minimum n = 4, 20 ROIs; R² = 0.9986. Inset, Raw SMLM image of TSPAN‐enriched EVs. The arrow represents the dilution at which the SMLM image was acquired. (c) Number of detected EVs per ROI in the CD9‐, CD63‐ and CD81‐enriched EV subpopulations and controls: anti‐rabbit IgG and anti‐cytochrome C Abs were immobilized onto surfaces; anti‐TSPAN Abs were immobilized onto surfaces and EVs were lysed with Triton X‐100; surfaces were not immobilized with Abs. Mean ± SEM; n = 4, 20 ROIs. (d) Raw SMLM images of CD9‐, CD63‐ and CD81‐enriched EVs. (e) 2D histograms and corresponding box plots for CD9‐, CD63‐ and CD81‐enriched EVs. Each dot represents a single EV with a corresponding diameter (x‐axis) and the number of detected TSPAN molecules (y‐axis). Box plots: interquartile range (box), median (centre line), mean (cross); the hollow dots indicate EVs detected beyond 1.5‐times the interquartile range (marked by the Whisker lines under and over the box). n = 4, 20 ROIs. ** Indicates p < 0.01; *** indicates p < 0.001. Numerical values and p‐values are provided in Tables S1 and S2.