nnLNSCs drive MDPs into a distinct macrophage population. (A) MDPs were isolated from BM cells of CX3CR1^+/gfp C57BL/6 mice by FACS. Numbers adjacent to gated areas indicate the percentage of cells before and after sorting. (B) Flow cytometry analysis of nnLNSCs purified by MACS. CD45.2 versus CD45.1 and gp38 versus CD31 expression of nnLNSCs after MACS-purification. (C) MDPs were cultured under the conditions indicated. After 5 days of culture the number of CD45⁺ cells has been determined. Fold changes are shown (mean ± SEM of 3 wells from 1 representative of 3 independent experiments). (D-I) MDPs cultured with 30 ng/mL GM-CSF only or on a monolayer of nnLNSCs for 10 days. MDPs (10⁴⁾ were seeded/well in 12-well plates and incubated. (D) At day 5, images were captured with bright-field or fluorescent microscope. Scale bar represents 100 μm. (E) At day 5, GFP-expression was monitored by flow cytometry (indicative for CX3CR1 expression), cells were gated on the DAPI⁻CD45⁺ population. (F) Cytospin of CD45⁺ cells sorted by MACS and stained with May-Grünwald-Giemsa after 5 or 10 days of coculture. Images were captured with bright-field microscope, original magnification 40×. (G) At day 5, the phagocytic ability of MDPs cultured with GM-CSF and MDPs educated by nnLNSCs was assessed by flow cytometry after OVA-Cy5 phagocytosis. Black, MDPs cultured with GM-CSF; red, MDPs educated with nnLNSCs (incubation at 37°C, solid line; incubation at 4°C, dotted line; without OVA-Cy5 control, shaded areas). (H-I) Analysis of CD45⁺ cells after 5 days of culture using antibodies as indicated. Data are representative of at least 3 independent experiments.