CD24 expression in murine and human dermal LECs in vitro and in vivo. (A) RNA expression in FPKM in three donors of human dermal LECs subjected to laminar (lam) shear stress, oscillatory (osc) shear stress, or static conditions (data from the bulk RNA-seq). (B) CD24 is not expressed at the protein level in in vitro–cultured human dermal LECs as assessed by flow cytometry. Representative flow cytometry plot of donor 431Z006.2. Donors 433Z032.3 and 439Z007.2 neither expressed CD24. (C) CD24 expression in mouse dermal imLECs. Representative flow cytometry plot of three experiments. (D and E) Whole mounts of Prox1-eGFP mouse ear showing expression of (D) CD24 in lymphatic valves visualized with Prox1 (high in valves) and (E) in combination with the cell adhesion molecule CD31. Representative images from five independent experiments. Scale bars: (D) 100 µm, (E) 25 µm. (F and G) Quantification of ITGA9⁺ valves was performed in 6-image Tilescans acquired in an ear skin area containing predominantly collectors (coll: CD31⁺aSMA⁺) or pre-collectors (precoll: CD31⁺aSMA⁻). (F) Representative image of a Tilescan (left) and of ITAGA9⁺ valves in CD31⁺aSMA⁻ pre-collecting (middle) and CD31⁺aSMA⁺ collecting vessels (right). Scale bars from left to right: 150, 100, 50 µm. (G) Quantifications of valves in the pre-collector or collector area of ear skin whole mounts from adult WT (Cd24^+/+) and Cd24^−/− mice (n = 8 WT and n = 7 Cd24^−/−). (H and I) Lymphatic drainage assay: adult WT and Cd24^−/− mice were injected with a near-infrared dye conjugate intradermally in the ear skin. Clearance of the tracer was monitored over 24 h by IVIS imaging. (H and I) (H) Average dye clearance plots and (I) calculated half-lives in WT and Cd24^−/− mice (data from one experiment with four to five mice per group are shown). ns, not significant; FPKM, fragments per kilobase of transcript per million mapped reads.