Expression of eNOS in endothelium-adherent monocytes in co-culture.

Dual-colour flow cytometry analysis of CD14 and eNOS expression on freshly-isolated PBMCs before co-culture (representative of 2 individual experiments) is shown (A). HLA-A2+ PBMCs (1×10⁶ cells/well) were incubated for 2 h with HLA-A2− HUVECs, after which the non-adherent PBMCs were removed by washing. (B) Shows the flow cytometry analysis of the cell layers for HLA-A2 and eNOS expression on Day 1 of co-culture (representative of 3 individual experiments). (C) Shows the 2 parameters dot plot from 3-colour flow cytometry analysis on Day 1 of co-culture staining for HLA-A2, CD11b and eNOS. This plot is gated for HLA-A2+ cells (representative of 2 individual experiments). Immunofluorescent micrographs (×60 original magnification) showing endothelial-adherent HLA-A2+ cells (green) and eNOS expression (red) on Day 0 (D) and Day 1 (E) of co-culture (representative of 2 individual experiments). The nuclei were stained with DAPI. Scale bar = 20 µm.