Comparative analysis of angiogenic capabilities of human neutrophils, monocytes, and mature and polarized macrophages. (A) Angiogenic potential of intact leukocytes in the in vivo CAM angiogenesis model. (Upper) Freshly purified neutrophils and monocytes, and macrophages of M0, M1, and M2 phenotypes were incorporated into 2-tier gridded 3D collagen onplants (3 × 10⁴cells/onplant), which were grafted on the CAM of chick embryos. Images were acquired using an Olympus CKX-41 microscope equipped with Olympus U-LS30-3 video camera and Infinity Capture software and processed using Adobe Photoshop. Objective lens, ×20. (Lower) After 76 hours of incubation, the onplants were scored for the presence of newly developed angiogenic vessels distinctly localized above the plane of the lower mesh grids (yellow arrowheads). Images were acquired using an Olympus binocular microscope SZ-PT (Olympus America, Melville, NY) equipped with a DVC video camera and high-performance ImageJ plugin acquisition software. Objective lens, 10×. (B) Angiogenesis-inducing capability of intact cells. The levels of angiogenesis were quantified for individual onplants as a ratio of grids containing new blood vessels to total number of scored grids, providing an angiogenic index. From 4 to 6 embryos, each grafted with 6 collagen onplants, were analyzed in 3 independent experiments. Scattergram presents fold changes in the levels of angiogenesis compared with negative no-cell (NC) control (1.0). Lines represent means of fold changes in angiogenic indices. **P < .01; ***P < .0001. (C) Comparative analysis of mature and polarized macrophages in the mouse angiotube model. Intact viable M0, M1, and M2 macrophages were incorporated into 2.5 mg/mL native collagen at 2 × 10⁶ cells/mL. Phosphate-buffered saline (PBS) was used as a negative, no-cell control (NC). Collagen mixtures were polymerized in the ∼1-cm-long tubes (angiotubes), which were surgically implanted under the skin of immunodeficient mice. The angiotubes were excised from the mice 12 to 14 days after implantation (images on the top). The contents of angiotubes were lysed in equal volumes of lysing buffer and hemoglobin concentration was determined, providing measurements of angiogenesis-inducing capacity of tested cells. Scattergram presents fold changes in the levels of angiogenesis induced by different macrophage types compared with negative NC control (1.0). Lines represent means of fold changes. Shown is a representative experiment from 2 independent experiments, each using from 4 to 5 mice, each implanted with 4 angiotubes. **P < .01; ***P < .0001, between the means from the experimental groups vs NC control.