nnLNSCs induce maDCs into a similar macrophage population. (A) maDCs expressing high levels of MHCII and CD11c from either EGFP-C57BL/6 or CD45.1 C57BL/6 mice were sorted by FACS. Representative plots show the expression levels of MHCII and CD11c presort and postsort. (B) nnLNSCs promote maDCs proliferation. Continued proliferation of maDCs after being seeded onto nnLNSCs. EGFP-maDCs cultured alone or on monolayers of nnLNSCs. At the time points indicated images were captured with a fluorescent microscope, original magnification 20×. (C-E) Characteristics of maDCs educated by nnLNSCs. (C) Cytospins of CD45⁺ cells sorted by MACS from maDCs alone, or maDCs cultured on nnLNSCs for 10 days were stained with May-Grünwald-Giemsa, original magnification 40×. Black arrows indicate proliferating cells with double nuclei. (D) Phagocytic ability of maDCs and maDCs educated by nnLNSCs for 10 days assessed by flow cytometry by uptake of OVA-Cy5. Black lines, maDCs; red lines, maDCs educated by nnLNSCs (solid lines, incubation temperature: 37°C; dotted line, 4°C). (E) CD45.1⁺ maDCs (black lines) or maDCs educated by nnLNSCs for 10 days (red lines) were stained with antibodies indicated. Dotted lines, corresponding isotype controls. (D-E) Histograms are gated on CD45.1⁺ cells. Representative data from at least 3 independent experiments are shown.