Interstitial and intralymphatic DC migration is ROCK-dependent. (A-B). FITC painting experiments were performed in the ears of WT mice treated with Y27632 (Y27) of with PBS vehicle control. Twelve hours after FITC application, mice were killed and the ear-draining auricular LNs harvested. (A) LN single cell suspensions were analyzed by FACS for the presence of I-A/I-E⁺CD11c⁺FITC⁺ cells. The number within each FACS plot represents the percentage of gated cells. (B) Y27632 treatment significantly reduced DC migration, as demonstrated by the quantification of total numbers of I-A/I-E⁺CD11c⁺FITC⁺ cells. Data from 1 of 5 similar experiments are shown. (C) Schematic representation of IVM experiments: YFP⁺ BM-DCs were injected into the ear skin of VE-cadherin-Cre × RFP mice, and baseline DC migration was recorded after 4 hours (2 × 1 hour). Two hours later, mice received intraperitoneal injections of Y27632 (10 mg/kg) or of PBS vehicle control. Imaging (2 × 1 hour) was initiated 3 hours after treatment. (D) Representative images of DCs present in the interstitium. Scale bar: 20 μm. (E) Y27632 treatment induced a significant elongation of interstitially migrating DCs. Quantification of the maximal DC length is shown. (F) Y27632 treatment induced a significant increase in the DC axis ratio, which was calculated by dividing the cellular length (l) by the cellular width (w). (G) Quantification of the effect of Y27632-treatment on the velocity of DC migration in the interstitium. (H) Quantification of the effect of Y27632-treatment on the velocity of intralymphatic DC migration. Dots represent individual cells. Pooled data from 6 (baseline) and 3 (PBS/Y27) different experiments are shown for panels E through G. Panel H shows pooled data from 5 (baseline), 3 (PBS), and 4 (Y27) different experiments. Baseline: before treatment; PBS: 3 to 4 hours after treatment with PBS; and Y27632: 3 to 4 hours after treatment with Y27632 (*P < .05; **P < .01; ***P < .001).