Fig 1: Purity of EV samples assessed by particle/protein ratio and Western blotting. (a)‐(c) The protein concentrations of EV preparations separated by different methods from CCM, urine and plasma. The protein concentrations have been corrected for initial sample input volume. (d)‐(f) The particle/protein ratios of EV preparations separated by different methods from CCM, urine and plasma. The particle numbers were measured by nFCM. The error bars represented the standard deviation of three repetitive experiments. *P < 0.05, **P < 0.01, one‐way ANOVA analysis with Tukey multiple comparison test as well as a variance‐covariance model. (g) Western blots of flotillin‐1, CD63, CD81, and calnexin for EV preparations separated from CCM by different methods. 9 μg of protein from each EV preparation was loaded. (h) Western blots of flotillin‐1, CD63, CD81, THP and calnexin for EV preparations separated from urine by different methods. 3.3 μg of protein from each EV preparation was loaded. (i) Western blots of flotillin‐1, CD81, APOA1 and calnexin for EV preparations separated from plasma by different methods. 25 μg of protein from each EV preparation was loaded. MCF7 membrane and cytosolic protein fractions served as positive and negative controls for flotillin‐1, CD63, CD81 and calnexin. Whole urine sample was used as positive control and MCF7 cellular fractions as negative control for THP. Recombinant human Apolipoprotein A1 served as positive control for APOA1.GAPDH staining was to show proper loading and running of the cytosolic MCF7 fraction
Fig 2: Biological characterisation of crude and purified EVs. Characterisation of particle size distributions by NTA in crude (n = 20, a) and purified samples (n = 5, b). Data are displayed as boxplots from the 10th to the 90th percentile, showing median (line) and mean (+) particle diameters. Median diameters of crude EVs from arterial and venous serum did not differ significantly (p = 0.056), while mean diameters of arterial particles were significantly smaller (p = 0.040). Particle diameters in additionally purified samples were also significantly smaller for arterial samples (median: p = 0.034, mean: p = 0.031). Depiction of particle concentrations/ml serum by NTA in crude (n = 20, c) and purified samples (n = 5, d). No significant changes in particle numbers could be detected for arterial and venous crude (p = 0.059) and purified EVs (p = 0.650). Lines indicate mean. Illustration of spherical EV-like extracellular particles with a size of about 100 nm by TEM for crude (e) and purified samples (f). Matched specimens from n = 3 individuals were imaged for both isolation protocols. Scale bar 1st and 3rd column: 250 nm; Scale bar 2nd and 4th column: 100 nm. Immunoblot profiling of SEC-purified samples (g). Arterial and venous samples were positive for EV-specific protein markers (alix, CD63, syntenin and CD81). CNX could not be detected in any of the samples. All samples were positive for non-EV-enriched proteins ApoA1 and serum albumin. Results are depicted for three paired arterial and venous biological replicates. HEK293 cell lysate was used as a positive control for alix, CD63, syntenin, CD81 and CNX. Recombinant ApoA1 and HepG2 cell lysate served as a positive control for ApoA1 and serum albumin, respectively.
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