Absence of TLR triggering or of commensal flora moderately diminishes the percentage of ALDH⁺ DCs. Light-density cells isolated from spleen (SPL) and from MLNs and CLNs of MyD88^−/−Trif^Lps2/Lps2 double-deficient mice and from wild-type C57BL/6 mice kept under SPF or germ-free conditions were incubated with ALDEFLUOR in the absence or presence of DEAB (not shown) and analyzed by flow cytometry for expression of CD11c, MHCII, CD11b, and CD103 and for ALDH activity. (A-B) The percentage of ALDH⁺ cells within mDCs from MLNs, CLNs, and cDCs from spleen is indicated. Data correspond to mean ± SD of at least 2 separate experiments involving 3 separate animals. Statistical comparison of the values corresponding to wild-type SPF mice with germ-free or MyD88^−/−Trif ^Lps2/Lps2 double-deficient mice. *P < .05. (C) Expression of CD103 and CD11b on total and on ALDH⁺ mDCs from MLNs and CLNs isolated from wild-type and MyD88^−/−Trif^Lps2/Lps2 double-deficient mice. (D) Expression of CD103 and CD11b on total and on ALDH⁺ mDCs from MLNs and CLNs isolated from SPF and germ-free mice. In panels C and D, numbers in outlined areas indicate percentage of cells, and FACS profiles are representative of 1 of 3 individual animals from at least 2 separate experiments.