Monocyte subset-specific identifiers. (A) Surface expression of distinct markers on CD14⁺⁺CD16⁻ monocytes (blue columns), CD14⁺⁺CD16⁺ monocytes (red columns), and CD14⁺CD16⁺⁺ monocytes (green columns) performed by flow cytometry. Data were measured as median fluorescence intensity (MFI) and presented as means ± SEM. Background fluorescence (measured in negative controls) was subtracted. Statistical analysis was performed using the Kruskal-Wallis test. (B) NK cells and neutrophil-depleted PBMCs before (left dot plot) and after (right dot plot) incubation with anti–HLA-DR MicroBeads and subsequent negative isolation. (C) Flow cytometric analysis of spontaneous intracellular ROS levels within the 3 monocyte subsets using the ROS-detection reagent carboxy-H₂DFFDA. Data are presented and analyzed as described in panel A. (D) CD4⁺ T-cell proliferation, measured flow-cytometrically as cytoplasmic dilution of CFDA-SE. Monocyte subsets were isolated, stimulated with SEB (2.5 μg/mL), and cultivated with CFDA-SE–labeled CD4⁺ T cells for 3 days. After gating for CD3-positive cells, percentages of proliferating CD4⁺ T cells were determined and denoted as means ± SD. Representative examples of 5 independent experiments are shown. (E) Stimulation of isolated CD14⁺⁺CD16⁻ monocytes with 2.5 μg/mL SEB versus control. After 24, 48, and 72 hours, percentages of CD14⁺⁺CD16⁺ monocytes (left panels) and expression of HLA-DR (right panel) of total events was determined flow-cytometrically. Percentages of CD14⁺⁺CD16⁺ monocytes derived from CD14⁺⁺CD16⁻ monocytes are given as means ± SD. Representative examples of 5 independent experiments are shown. HLA-DR MFI was measured as described in panel A. Red arrowhead marks HLA-DR expression of unstimulated CD14⁺⁺CD16⁺ monocytes (compare panel A). HLA-DR MFI of SEB-stimulated and control cells were compared by the paired Student t test; *P < .05, **P < .01. (F) Bottom panel: Surface expression of KDR (VEGFR2) on monocyte subsets measured by flow cytometry. Data are presented and analyzed as described in panel A. Top panel: Monocyte subsets cultivated for 3 days on Matrigel in the presence of 10 ng/mL VEGF (RPM1 medium/5% FCS). Representative examples of 3 independent experiments are shown. HUVECs were used as control cells (EGM-2 medium/5% FCS). Image acquistion was performed by the Keyence BZ-8000K microscope equipped with a Nikon Plan Apo 4×/0.2 objective and the BZ Viewer software, magnification 8-12×, room temperature. (G) Capacity to phagocyte opsonized carboxylate microspheres (0.75 μm, Yellow Green) by the 3 monocyte subsets within 30 minutes; counts of FITC-positive cells were determined flow-cytometrically and are denoted as means ± SD. Representative examples of 10 independent experiments are shown.