DCs actively crawl inside initial lymphatics using lymph flow as a directional cue. (a) Sequential images of BMDC propagation inside initial lymphatics. Circled is a DC crawling inside the lymphatic lumen. (b) At a higher magnification a DC is seen crawling using lamellipodia at the leading edge and a well defined uropod at the trailing edge. Sequences are representative of at least 13 independent experiments. (c) Under general anesthesia, which is known to reduce lymph flow, the percentage of immigrant GFP⁺ BMDCs from the footpad skin, as recorded in the popliteal LN 10 h after transfer, drops fivefold (P < 0.001). Data represent three independent experiments. Red bars denote the mean. *, P < 0.001. (d–f) In the presence of lymph flow, DCs move directionally in the lymphatics. Mice were injected s.c. with 5 µl of fluorescently stained saline (red). As fluid diffused through the interstitium and concentrated in lymphatics, DCs switched from random motility inside lymphatics (d) to directional crawling toward the LN (e). Tracks show several DCs following the same route down the lymphatic vessel (f). Under these conditions DCs crawled along the lymphatic vessel at a mean speed of 9.06 µm/min and a persistence index of 0.65.