c-FLIP^f/f LysM-Cre mice lack marginal zone macrophages and have splenomegaly and splenic extramedullary hematopoiesis. (A) Spleen weight (left) and cellularity of spleens lysed of red blood cells (right) in c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice. Black boxes represent c-FLIP^f/f mice and white boxes represent c-FLIP^f/f LysM-Cre mice. Data were obtained in 5 independent experiments (spleen weights) or 7 independent experiments (splenic cellularity). **P < .01; ***P < .001. (B) Loss of CD115⁺ ER-TR9⁺ marginal zone macrophages but not MOMA-1⁺ metallophilic macrophages as observed by immunofluorescent staining. Frozen spleen sections from c-FLIP^f/f (left) and c-FLIP^f/f LysM-Cre (right) mice were stained for CD115 (red) and B220 (green; top), or MOMA-1 (blue) and ER-TR9 (green; bottom). Original magnifications 100× (top), 200× (bottom). Images were obtained using a Zeiss Axiovert 200M (10×/0.30 NA objective lens, top; 20×/0.75 NA objective lens, bottom) with an AxioCam MRm camera and AxioVision Rel. 4.8 software. (C) Images of paraffin-embedded spleen sections from c-FLIP^f/f and c-FLIP^f/f LysM-Cre mice stained with H&E. Original magnification 100×. Inset, image showing megakaryocytes in the spleen of a c-FLIP^f/f LysM-Cre mouse. Original magnification 400×. Images were obtained using a Zeiss Axiovert 200 (10×/0.25 NA objective lens or 40×/0.50 NA objective lens, inset) with an AxioCam MRC camera and AxioVision Rel. 4.8 software. (D) Absolute numbers of CD4⁺ or CD8⁺ T cells, B220⁺ B cells, CD11b⁺ Ly6C^int neutrophils, and total SSC^lo cells (left) or SSC^lo CD11b⁺ Ly6C^hi inflammatory monocytes and SSC^lo CD11b⁺ Ly6C^lo resident monocytes (right) in c-FLIP^f/f and c-FLIP^f/f LysM-Cre spleens as determined by flow cytometry. Data were obtained in 4-7 independent experiments (T cells, 5 experiments; B cells, 4 experiments; neutrophils and monocytes, 6 experiments, and SSC^lo cells, 7 experiments). n = 7-14; NS, not significant; ***P < .001. (E-G) Absolute numbers of progenitor cells in spleen as determined by flow cytometry. Data were obtained in 2 independent experiments. n = 4; *P < .05; **P < .01. (E) Absolute numbers of Lin⁻ (CD3ϵ⁻CD4⁻CD8⁻CD11b⁻B220⁻) cells. (F) Absolute numbers of LSK (Lin⁻Sca-1⁺c-Kit⁺) cells, CMP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁺ FcγR^lo), and GMP (Lin⁻c-Kit⁺Sca-1⁻ CD34⁺ FcγR^hi) populations. (G) Absolute numbers of MEP (Lin⁻ c-Kit⁺ Sca-1⁻ CD34⁻ FcγR^lo) cells. In all panels, black bars represent c-FLIP^f/f mice and white bars represent c-FLIP^f/f LysM-Cre mice. *P < .05; **P < .01; ***P < .001. Error bars represent standard deviations.