R848 administration mobilizes CD11c/YFP⁺, but not CX3CR1/GFP⁺, cells into MLN-afferent lymph. (A) Visualization of MLN-afferent lymphatics. 1 h after gavage of 200 µl of olive oil, lymphatics appear as bright/white vessels running in parallel to mesenteric blood vessels (left). FITC- and TRITC-labeled dextran injected i.v. 2 and 30 min before analysis (green and red, respectively) can differentially highlight blood vessels or both blood vessels and lymphatics. In the merge (right) image, blood vessels appear yellow and lymph vessels red. Bar, 1 mm. (B) Two-photon microscopy of MLN-afferent lymphatics of CD11c-EYFP and CX3CR1^+/GFP mice in the steady state or 5 h after oral administration of R848. TRITC-labeled dextran was injected i.v. 30 min before mice were sacrificed to visualize the vessel lumen (red). Collagen-rich vessel walls display a second harmonic signal (blue). The images show orthogonal projections of 20-µm-thick (CD11c-EYFP mice) or 5-µm-thick (CX3CR1/GFP mice) volumes. Bars, 50 µm. CD11c-positive cells were readily detectable in steady-state lymphatics (top left, green) and in increased numbers 5 h after gavage of 10 µg R848 (bottom left). CX3CR1-positive cells typically occurred in the shape of stellate morphology cells on the outside of vessel tubes or within the vessel wall (top right). Cells not expressing the reporter GFP/YFP appeared as TRITC-excluding shadows in the lumen. Pictures shown are from non–Peyer's Patch draining lymphatics. The following numbers of independent experiments were each performed with single mice: CD11c-EYFP, five; CD11c-EYFP⁺R848, two; CX3CR1^+/GFP, four; and CX3CR1^+/GFP+R848, five.