Impact of plasmin and plasmin(ogen) on in vitro DC migration. In vitro experiments were performed with LPS-matured bone marrow–derived DCs and primary LN LECs. (A) DC displayed greater chemotaxis toward CCL21-ΔC as compared with full-length CCL21. (B) DCs were allowed to transmigrate for 4 h across primary LN LEC monolayers. DCs displayed a near-significant in transmigration (i.e., in two out of three experiments) toward CCL21-ΔC compared with CCL21 added to the lower well compartment. (C and D) The presence of plasminogen (Plg) or plasmin (Plm), which were added to the upper and lower wells of the Transwell plate, did not impact DC chemotaxis toward (C) CCL21-ΔC and (D) CXCL12. Each data point represents an independent experiment. (E and F) The presence of plasminogen or plasmin in the assay (upper and lower wells) did not impact DC transmigration across LEC monolayers toward (E) CCL21-ΔC or (F) CXCL12. Each data point represents an independent experiment. (G and H) Crawling assay: YFP-expressing DCs were added on top of LEC monolayers, and their migration was recorded by time-lapse microscopy. The presence of plasminogen or plasmin in the medium did not impact the (G) velocity and (H) chemotactic index of DC crawling. Of note: Inhibition of ROCK with Y27632 was performed as positive CTR (Nitschke et al., 2012). Statistics: two-way ANOVA using multiple comparisons, followed by a Tukey correction. (I) qPCR-results demonstrating that in vitro–cultured primary LN LECs no longer express CCL21 and also do not express CCL19. S18: housekeeping gene; gp38: podoplanin. Raw CT values are shown. n = 3 different biological replicates (isolations).