Identification of ALDH⁺ DCs in the skin, lungs, and corresponding draining lymph nodes. Light-density cells isolated from the CLNs (A), the ear epidermis (B), the ear dermis (C), the MELNs (D), and the lungs (E) were incubated with ALDEFLUOR and analyzed by flow cytometry for expression of CD11c, MHCII, CD11b, CD103, CD45, and CD24 and for ALDH activity. Dot plots correspond to nonautofluorescent cells (skin and LNs) or to both autofluorescent and nonautofluorescent cells (lungs) with a FSC^high SSC^high profile characteristic of DCs and from which neutrophils were gated out using Ly6G and Gr-1 staining. Positioning of the ALDH⁺ gate is based on incubation in the presence of DEAB (supplemental Figure 3). For the CLNs and MELNs, the CD11c^{inter to high}MHCII^high and the CD11c^highMHCII^inter gates correspond to mDCs and lymphoid tissue–resident DCs, respectively. The gates corresponding to epidermal Langerhans cells, DDCs, alveolar MFs, and lung DCs are specified in supplemental Figure 2B through D. Numbers in outlined areas indicate percentage of cells. Data are representative of 3 separate experiments involving groups of 3 to 6 mice.