Effect of S100A9 overexpression on myeloid cell differentiation in transgenic mice. (A) Vector used for generation of transgenic mice. (B, left) GFP⁺ cells in peripheral blood of wild-type (WT) or transgenic mice (Tg) that express S100A9. (right) The level of S100A9 protein in spleens of wild-type (WT) and S100A9 transgenic mice (Tg). (C) Bone marrow progenitor cells were enriched by lineage depletion kit (MACS), and then stained with a cocktail of lineage-specific antibodies. Lin⁻c-kit⁺ GFP⁻ or GFP⁺ populations were sorted by flow cytometry. (D) Sorted cells were cultured for 5 d with cocktail of cytokines as described in Materials and methods. After that time, media was replaced and 0.5 × 10⁶ cells were plated in 24-well plates and cultured with GM-CSF alone for additional 7 d. LPS was added for the last 24 h of culture. The phenotype of cells was evaluated using multicolor flow cytometry. WT, wild-type FVB/N mice; GFP⁻ and GFP⁺, S100A9 Tg mice. Three experiments were performed. (E) Sorted cells were used as stimulators of allogeneic (BALB/c) T cells. Cell proliferation was measured in triplicate 4-d cultures by ³[H]thymidine uptake. Mean ± SD from two experiments are shown. T cells alone showed count <500 CPM.