Fig 2: Effect of AA on the release of EVs. (A) Relative number of particles released by MDMs into the culture medium after 24 h of treatment with AA (blue dots) or solvent (grey dots) determined by HSFC. Values were calculated per mg total cell protein and normalized to 1 for solvent-treated cells. (B) Size distribution of particles released by MDMs shown in panel A. Histogram of particle size with a bin width of 10 nm for AA (blue bars) and solvent control (grey bars) samples. Data were normalized to the total number of acquired events separately for solvent and AA-treated samples. The plot shows the mean (± SEM) of the relative frequency of total events detected by HSFC in conditioned media from n = 5 donors. (C) Relative decrease of particle count in conditioned media after treatment with Triton X-100 (final concentration 0.05%). Values obtained before addition of detergent were set to 100%. (D) Immunostaining results on EV pellets obtained by differential centrifugation. The presence of tetraspanin markers (CD9, CD63 and CD81) was evaluated on particles obtained from n = 3-5 donors. A histogram of particle size of positive events is shown in Figure S8 for a representative sample. Bars represent mean (± SD) of total % of positive events. Symbols represent the values obtained with independent donors. Significance was tested by paired t test: * p < 0.05 for AA versus solvent.

Fig 3: CB- and AT-MSC-derived EVs display a distinct surface marker profile. Detection of the surface marker proteins CD9, CD63, CD73, CD81, HLA-ABC, and HLA-DR using flow cytometry on EV preparations. The data are presented as means ± SD of normalized mean fluorescence intensities (MFIs), which were calculated as the ratio of the geometric MFI of EV samples (beads + EVs + antibodies) to control samples (beads + antibodies). Statistical analysis was performed by the Mann-Whitney test with * p < 0.05. ND indicates not detected. EVs from four different donors per cell type were included.