Fig. 3: Characterization of macrophages differentiated from the trilineage monocyte progenitor (TMP) isolated from peripheral blood mononuclear cells (PBMCs) of control subjects.

Using flow cytometry, TMPs were identified as CD15⁻CD45⁺CD3⁻CD19⁻CD56⁻CD11b⁺CD14⁺ cells, sorted, and differentiated into macrophages by adding M-CSF in wells coated with IgG anti-Fc only (IgG), or with additional treatment by Notch-ligand:Fc fusion proteins (JAG1 or DLL1). LPS and IFNγ were added 24 h before harvesting to induce the inflammatory response. A Gene expression in macrophages differentiated from TMPs was determined using TaqMan assays for macrophage-specific genes: CSF1R, NFKB1, STAT1, TNF, and CCL2, relative to the HMBS housekeeping gene and presented as normalized to the appropriate control wells (IgG anti-Fc without Notch-ligands) (n = 6 individual samples, 6 wells/sample). B The phenotype of macrophages differentiated from TMPs was determined using monoclonal antibodies specific for the macrophage lineage markers: CD163, CD1c, HLA-DR, CD206, and CD64 (n = 7 individual samples, 3 wells/sample). C Representative images and quantification of macrophage phagocytosis. Activation of rhodamine in acidic vesicles after phagocytosis was detected as pHrodo⁺ macrophages (magnification ×100). D Number and area × mean fluorescence intensity (MFI) of pHrodo⁺ macrophages were expressed per field of view and normalized to the appropriate control wells (IgG anti-Fc without Notch-ligands) (n = 4 individual samples, 2–3 wells/sample). Box-and-whisker plots: horizontal lines represent the median, boxes represent the interquartile range (IQR), and whiskers represent 1.5 times the IQR. Statistically significant difference was determined at p <0.05, Kruskal–Wallis with Conover post-hoc test.