Endothelial cells induce differentiation and expansion of macrophages in vitro. (A) Schematic representation of the coculture system that includes murine hematopoietic cells (HCs) and immortalized mouse endothelial cells (IMECs). All HCs express DsRed fluorescent protein. (B) Phase contrast (left) and fluorescent (right) micrographs depict the formation of DsRed⁺ colonies (arrow) during coculture. Note that the colonies were phase-dim or invisible under phase view. Scale bar, 200 μm. (C) Photomicrographs of isolated bone marrow (BM) cells and cells from day 27 colonies (CC, arrow) after May-Grünwald-Giemsa staining. Scale bar, 50 μm. (D) FACS profile (left) indicates that the large majority of cells in the colonies were F4/80⁺/Mac-1⁺ (n = 3). (E) Fluorescent micrographs of bromodeoxyuridine (BrdU) staining indicating active proliferation within the colony. Day 10 colonies were pulse-labeled with BrdU for 8 hours and then costained for anti-BrdU (red) and anti-F4/80 (green) antibodies. Arrow, cells positive for both F4/80 and BrdU. Arrowhead indicates F4/80⁺ cells not stained for BrdU. DAPI indicates staining for nucleus (blue). Scale bar, 50 μm. (F) Colony cells uptake DiI-ac-LDL. Cells were incubated with 10 μg/mL DiI-ac-LDL for 4 hours, followed by fluorescent microscopy (left). (Right) FACS profile shows the majority colony cells take up Dil-ac-LDL. CD31, to exclude possible IMEC contamination. Scale bar, 200 μm.