Fig. 5

CardAP-EVs prime CD14⁺ monocytes in unstimulated PBMC cultures towards a regulatory CD14⁺ myeloid cell type. 1x10⁶ PBMCs were treated with DiD-labelled CardAP-EVs for 24 h and analysed by microscopy or flow cytometry. a Representative images are illustrating co-localization (white arrows) of DiD⁺EVs (magenta) with CD14⁺ PBMCs (green) in total PBMCs (pseudo coloured white for DAPI) (n = 2; three different CardAP donors, two different PBMC donors). Scale bars represent 10 µm. b Representative histograms of the flow cytometric analysis are shown for CD14⁺ and CD14⁻ immune cells (n = 2; three CardAP donors; two different PBMC donors). For the phenotypical analysis, 1x10⁶ PBMCs were treated with unstimulated (EVs) or cytokine stimulated EVs (EVs^(cyt)), PBS in equal volume of the EVs (PBS) or left untreated. After 3 days, cells were analysed by flow cytometry. c Frequencies of CD14⁺ cells in PBMCs are presented for cultures treated with PBS, EVs or EVs^(cyt). d Flow cytometric surface expression data are presented as median with interquartile range of normalized geometrical mean fluorescence intensities (normalized MFI calculated as ratio of stained to unstained) for the immunological markers HLA-DR, CD86, PD-L1 and CD206 (n = 11; four CardAP donors, four PBMC donors). Friedman Test with Dunn´s multiple comparison test: ***p < .001, **p < .01, *p < .05 or Wilcoxon matched-pairs signed rank test; ^###p < .001, ^##p < .01, ^#p < .05