Transitory macrophages enhance early states of fibroblast outgrowth during humanization. (A) Primary human monocytes/macrophages were isolated via Ficoll separation from apheresis product, differentiated, and then transduced with green fluorescent protein (GFP). Images show cells 48 hr post-transduction with GFP or negative controls. Cell nuclei were stained with diamidino-2-phenylindole (DAPI) for visualization (blue, bottom panels). (B) Primary breast fibroblasts were injected with or without the GFP-expressing macrophages in the presence or absence of ectopic estrogen. Six days post-injection freshly excised glands were imaged using a GFP filter. Immunohistochemical detection of human macrophages (day six) humanized glands using a human-specific CD14 antibody (top panels) or corresponding negative controls (bottom panels; scale bar = 200 μm). (C) Percent gland humanized after ten days of fibroblast growth +/- estrogen +/- macrophages. Total and humanized areas were determined by histological examination, imaged with the Zeiss SteREO Discovery V12 and calculated using Axiovision V4.8 software. Data represent percent humanization ± SD. Four glands per treatment group were measured. *P < 0.01. (D) Tissues were subjected to immunohistochemical analysis with a proliferating cell nuclear antigen (PCNA) antibody to identify proliferative cells. Stained histosections were imaged and quantified using NIH Image J software. Data represent average of four counts per gland ± SD. Three glands per treatment group were measured. *P < 0.02.