DC migration from skin into the LN parenchyma is enhanced in uPA ^mut mice. FITC was applied to the ear skin in WT and uPA^mut mice, and ear-draining auricular LNs were collected for analysis after 24 h. (A–D) Flow cytometry–based quantification of DCs in single-cell suspensions of enzymatically digested LNs. Percentage (A and C) and absolute numbers (B and D) of all migratory DCs (A and B; CD11c⁺MHCII⁺) and FITC⁺ migratory DCs (C and D). Pooled data from two similar experiments are shown (n = 16–19 mice per group). (E–G). Immunofluorescence-based analysis of sections prepared from auricular LNs after FITC painting. (E) Representative whole-slide multiplex immunofluorescence images of WT and uPA^mut LNs. Images on the far left: overall LN architecture and examples of regions of interest (yellow boxes) used for high-resolution analysis of the SCS and parenchymal compartments. Subsequent images (left to right): higher-magnification images showing a merge of FITC signal, CD11c (DCs) and LYVE-1 (LVs) staining, followed by single-channel views of FITC and CD11c. Images on the far right: AI-based segmentation maps of FITC⁺CD11c⁺ DCs in the SCS or parenchyma. Scale bar.: 100 μm. (F) Percentage of FITC⁺CD11c⁺ DCs localized in the SCS or in the LN parenchyma, and (G) ratio of FITC⁺CD11c⁺ DCs in the SCS vs. LN parenchyma in WT and uPA^mut LNs. Pooled data from the analysis of 6–7 mice per genotype are shown. Each dot presents the average of 3–6 images analyzed per one mouse. (H) Comparison of the LN cellularity retrieved from enzymatically digested (“D”) or nondigested (“ND”) LNs. Data points belong to the experiments described in A–D; “D” and I–L, “ND”. (I–L) Flow cytometry–based quantification of DCs in single-cell suspensions generated not digested LNs. Percentage and absolute numbers of (I and J) all migratory DCs (CD11c⁺MHCII⁺) and (K and L) FITC⁺ migratory DCs. Pooled data from three similar experiments are shown (n = 31–34 mice per group). Unpaired Student’s t test (all graphs).