BM cells from femurs of CCR7+/+ or CCR7−/− mice were cultured in RPMI 1640 (GIBCO BRL) supplemented with 10% FCS (HyClone) and recombinant mouse GM-CSF (R&D Systems) according to current protocols. To induce maturation, 8–10 d BM-DC cultures were stimulated with 0.5 μg/ml LPS (Sigma-Aldrich) for 24 h. At that time point DCs were homogeneously CCR7+ as detected by staining with a CCL21–Ig fusion protein (provided by K. Karjalainen, Institute for Research in Biomedicine, Bellinzona, Switzerland). CD11c+ and CD11c− spleen cells were isolated using CD11c-coated magnetic beads (Miltenyi Biotec). Cells were labeled with 2.5 μM 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) or 10 μM 5- and 6-(4-chloromethyl)benzoyl-amino-tetramethylrhodamine (CMTMR) intracellular fluorescent dyes according to the manufacturer's instructions (Molecular Probes). After labeling, cells were washed extensively in PBS and injected s.c. into the footpad or flank (for experiments involving use of CFA or IFA). No differences in the extent of DC migration to lymph nodes were found when CFSE or CMTMR was used.