Characterization of macrophages in E9.5 to E11.5 MacGreen HBA. (A) Schematic diagram of dissected HBA region on the mouse embryonic head. The region includes the first and second branchial arches. (B) Flow cytometric profile for E10.5 MacGreen HBA cells showing that all GFP⁺ (high-expressing) cells are CD45⁺CD11b⁺F4/80⁺Gr1⁻ macrophages. Percentages shown in gated areas. FSC, forward scatter. (C) Percentages of MacGreen GFP⁺ cells in E9.5, E10.5, and E11.5 HBA (n ≥ 3). *P = .016; ***P < .001. (D) Three-dimensional whole-mount images of an immunostained MacGreen E10.5 head (34 somite pairs [sp]), with boxed areas enlarged in right panels. Anti-GFP (green) and anti-CD31 (magenta) antibody staining shows localization of macrophages surrounding the CD31⁺ vasculature. CA, carotid artery; NE, neuroepithelium; V, brain ventricle. Bar = 10 μm. (E) Representative flow cytometric data showing MFI and percentage of GFP⁺ macrophages and GFP⁻ cells expressing chemokine receptors in the E10.5 (32 to 39 sp) MacGreen HBA. Dotted line = FMO; blue line = GFP⁻ cells; gray filled = GFP⁺ cells. (F) Bar graphs showing percentages of chemokine receptor-expressing cells in the GFP⁺ fraction. n = 4 for Cx3cr1, Ccr7, Ccr5, Ccr3 and n = 3 for Cxcr4, Cxcr2.