Frequency and migration of DCs from ear skin in ICSBP^-/- mice. Ears from 3 to 5 ICSBP^-/- and WT mice were excised and split into dorsal and ventral halves. Dorsal halves were further treated, as indicated in “Materials and methods,” to allow the separation of dermis from epidermis. (A) Dermal and epidermal sheets were incubated ventral-side down in medium containing GM-CSF for 24 hours, then cells that migrated from the tissue into the culture medium were collected and double-stained for CD11c and I-A expression. R1 region indicates the percentage of double-positive cells within the total population of living cells; KO, knock-out. (B) Dermis and epidermis were incubated in presence or absence of 6Ckine. Forty-eight hours later, cells that migrated ex vivo were collected, counted, and stained for CD11c coupled with the indicated antibodies. The number of positive cells was calculated by applying the percentage of double positivity to the mean number of migrated cells. (C) Freshly isolated epidermal sheets were passed through a steel sieve and cell suspensions were centrifuged on a Nycodenz gradient. The low-density fraction was collected and stained for identification of LCs. Representative data of 1 of 3 experiments are shown.