Galectin-9 function in DC migration is conserved in vivo. (A) Scheme depicting experimental setup. WT and galectin-9^−/− BMDCs were stained with far-red or violet CFSE dyes, mixed in equal numbers, and injected into the footpad or tail vein of donor mice. 48 h later, draining lymph nodes were isolated and donor BMDCs enumerated by flow cytometry. (B) Representative flow cytometry plots depicting the cellular mixtures injected into recipient mice. In mixture A, WT BMDCs were stained with far-red CFSE and galectin-9^−/− cells received violet CFSE. For mixture B, colors were interchanged to discard any effect of the dye in cell migration. (C and D) DC mixtures from (B) were injected into WT (C) and galectin-9 KO (D) host mice. Data depict the percentage of migratory donor BMDCs ± SEM. Each symbol represents values obtained for one draining lymph node. Statistical significance was determined using one-way ANOVA followed by the Holm–Sidak multiple comparisons test. (E) Homing index of WT over galectin-9 KO DCs for each of the injected cell mixtures and host mouse genotype. The homing index was calculated using the following formula: (% far-red signal in tissue/% violet signal in tissue)/(% far-red signal in input/% violet signal in input). Each mixture was injected three times into either WT or gal9 KO host mice. The homing index is depicted. (F) BMDCs obtained in A were also subjected to a chemokine transwell assay in the presence or absence of chemokine CCL21. Data show the percentage of moDCs that migrated relative to input. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05; ***P < 0.0001.