Fig. 6.

IVIG-SiMPs increased the release of EVs positive for autophagosome and mitochondrial markers in TNFα-stimulated HUVECs. After exposure HUVECs to 200 nm IVIG-SiMPs (100 µg/ml) and the controls for 24 h. in the presence of 10 ng/ml TNFα, the released of EVs in the cell culture supernatant were collected and subjected to flow cytometry for evaluation of the EV size distribution and phenotyping. The EV surface expression of the following markers was analyzed: (A) CD105/PS for endothelial cell origin and procoagulant phenotype, (B) CD105/LC3 to detect the autophagosome marker, and (C) TOM20/LC3 to detect EVs of mitochondrial origin and products of mitophagy. (D) Flow cytometry analysis of EV size distribution was performed in comparison to the ApogeeMix reference silica beads. The overlayer (D, a) pseudo dot plot and (D, b) histogram plot of IVIG-SiMPs, bare SiMPs, and Apogee beads. (E) HUVECs were treated with IVIG-coated 200 nm blue, fluorescent SiMPs (100 µg/ml) to monitor the release of EVs associated with IVIG-SiMPs and positive for endothelial membrane (CD105) and autophagosome (LC3) markers. Mean ± SEM, n = 3, *, # p < 0.05, **, ## p < 0.01, ***, ### p < 0.001