Supplemental data to FITC painting experiments and analysis of LNs draining CHS-inflamed skin. (A) Schematic depiction of the experiment: FITC was applied to the ear skin in WT and uPA^mut mice, and ear skin or ear-draining auricular LNs were collected for analysis after 24 h. (B and C) Analysis of dermal DC numbers. Ear skin was enzymatically digested, and single-cell suspensions were generated for flow cytometry–based analysis. (D and E) Gatings used to identify migratory DCs (CD11c⁺ MHCII^hi), and, amongst those, FITC⁺ DCs in single-cell suspensions generated from (D) enzymatically digested LNs and (E) undigested LNs. (F–I) Analysis of immobilized CCL21 and plasmin(ogen) in CHS-dLNs. A CHS response was induced in the ear skin of WT mice, and ear-draining auricular LNs were collected 24 h later. (F and G) Analysis of CCL21 levels in LNs draining CTR or CHS-inflamed ear skin. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. (F) Representative images of the immunofluorescent staining and of the AI-based tissue segmentation used for differentiating between the T cell zone and B cell follicles/SCS. Scale bar: 100 μm. (G) Quantification of CCL21 staining intensity of observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. (H) ELISA-based quantification of (H) plasminogen and (I) plasmin activity in LN protein extracts, generated after perfusing the mice with PBS. Pooled data from n = 6 mice per group are shown in H and I, Student’s t test. (J) Representative gating strategy used for the quantification of FITC⁺CD11c⁺ cells in LN sections from WT and uPA^mut FITC-painted auricular LNs in Fig. 9, E–G. CD11c⁺ cells were identified based on CD11c-AF647 positivity. From the CD11c⁺ population, FITC⁺ cells were identified and subsequently quantified. Marker-negative cells (red color) served as negative control to check background staining and to set the fluorescence thresholds. Please note that the data points of the WT CTR group in C are identical to those shown in Fig. 8 F, as these extracts were prepared and measured simultaneously.