Figure 4. Membrane-associated... | Rockefeller University Press

Figure 4.

Membrane-associated macrophages originate from embryonic precursors. (A) Whole-mount images of mesentery in newborn (P0) CX3CR1gfp/+ pups stained with LYVE1. Right-most image is the enlargement of the boxed area in the adjacent image. Arrows indicate LYVE1lo/− CX3CR1-GFP+ macrophages. Images are representative of two independent experiments. Scale bar, 100 µm; 30 µm (higher-magnification image). (B) CX3CR1-GFP expression within the Lyve1hi macrophage pool in the mesenteric membrane of newborn mice. GFP expression is quantified within total LYVE1hi macrophage population. (C) The frequency of LYVE1hi macrophages versus LYVE1lo/− macrophages in the mesenteric membrane of newborn mice. In B and C, data are representative of two independent experiments (n = 4; mean ± SEM). Membrane-associated macrophages were quantified in one to three different regions of mesenteric membrane per mouse. Unpaired Student’s t test: ****, P < 0.0001. (D) Whole-mount images of MLNs and mesenteric membranes of P1, P6, and P14 neonatal mice stained with LYVE1 and MHCII. White, LYVE1; green, MHCII; blue, DAPI. The yellow line in the P1 panel indicates the border of mesenteric vessels and mesenteric membrane. Images are representative of at least two independent experiments per time point. Scale bar, 50 µm. (E) Representative whole-mount images of adult CX3CR1CreERT2:R26LSL-Tomato mice in which tamoxifen was injected on P1 (white, LYVE1; red, Tomato reporter). Scale bar, 50 µm. (F) Tomato expression in microglia, LYVE1hi mesenteric membrane–associated macrophages and blood monocyte subsets. LYVE1hi membrane-associated macrophages were quantified in two different regions of membrane per mouse from confocal microscopy images. Microglia and blood monocytes were quantified via flow cytometric analysis. In E and F, data are pooled from two independent experiments (n = 6; mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001.

Membrane-associated macrophages originate from embryonic precursors. (A) Whole-mount images of mesentery in newborn (P0) CX₃CR1^gfp/+ pups stained with LYVE1. Right-most image is the enlargement of the boxed area in the adjacent image. Arrows indicate LYVE1^lo/− CX₃CR1-GFP⁺ macrophages. Images are representative of two independent experiments. Scale bar, 100 µm; 30 µm (higher-magnification image). (B) CX₃CR1-GFP expression within the Lyve1^hi macrophage pool in the mesenteric membrane of newborn mice. GFP expression is quantified within total LYVE1^hi macrophage population. (C) The frequency of LYVE1^hi macrophages versus LYVE1^lo/− macrophages in the mesenteric membrane of newborn mice. In B and C, data are representative of two independent experiments (n = 4; mean ± SEM). Membrane-associated macrophages were quantified in one to three different regions of mesenteric membrane per mouse. Unpaired Student’s t test: ****, P < 0.0001. (D) Whole-mount images of MLNs and mesenteric membranes of P1, P6, and P14 neonatal mice stained with LYVE1 and MHCII. White, LYVE1; green, MHCII; blue, DAPI. The yellow line in the P1 panel indicates the border of mesenteric vessels and mesenteric membrane. Images are representative of at least two independent experiments per time point. Scale bar, 50 µm. (E) Representative whole-mount images of adult CX₃CR1^CreERT2:R26^LSL-Tomato mice in which tamoxifen was injected on P1 (white, LYVE1; red, Tomato reporter). Scale bar, 50 µm. (F) Tomato expression in microglia, LYVE1^hi mesenteric membrane–associated macrophages and blood monocyte subsets. LYVE1^hi membrane-associated macrophages were quantified in two different regions of membrane per mouse from confocal microscopy images. Microglia and blood monocytes were quantified via flow cytometric analysis. In E and F, data are pooled from two independent experiments (n = 6; mean ± SEM). Statistical analysis was performed by one-way ANOVA and Tukey’s multiple-comparison test: ****, P < 0.0001.