Endothelial cells impart M2 polarity on colony macrophages. (A) MHCII expression in colony cells is reduced on coculture, as determined by FACS overlay graph (left) and quantification (right; n = 3). (B) Tie2 expression in colony cells is gradually increased over time as shown by FACS overlay graph (left) and quantification (right; n = 3). (C) Colony cells exhibit M2 macrophage markers but not M1 markers, as detected by real-time PCR analysis. Ctrl indicates bone marrow–derived macrophages stimulated with 50 ng/mL LPS (for M1 control) or with 5 ng/mL IL-4 (for M2 control) for 24 hours (n = 6). (D) Colony cells express high level of Vegfa transcripts. (E) Immunolocalization of F4/80 (pan macrophage marker, red) and CD206/Mrc1 (M2 macrophage marker, green) in a macrophage colony. Note that cells in the center of the colony are F4/80⁺ and CD206⁻; cells on the periphery are F4/80⁻ and CD206⁺; cells in between are positive for both markers. Colony shown is from day 14 coculture. DAPI indicates staining for nucleus (blue). Scale bar, 50 μm (*P < .05; unpaired Student t test).