(A) Flow cytometric detection of GFP fluorescence in gated splenic CD11c+MHCII+ cells from Ebi2GFP/+ mice. (B) Quantitative PCR analysis of Ebi2 transcript abundance in sorted splenic CD11c+MHCII+CD4+ cells (CD4+ DCs) and CD11c+MHCII+CD8+ cells (CD8+ DCs). Expression is shown relative to Hprt (n = 4 mice). (C) EBI2 surface staining of gated splenic CD4+ and CD8+ DCs from Ebi2−/− or Ebi2+/+ mice (one representative of four experiments). (D) Migration of CD4+ DCs and CD8+ DCs in response to 7α,25-OHC (mean from four mice ± SE, combined from two experiments). (E)–(I) Flow cytometry and enumeration of splenocytes (E, F, and G), inguinal LN cells (H), and mesenteric LN cells (I) from Ebi2−/−, Ch25h−/−, Cyp7b1−/−, Hsd3b7−/− mice, and their matched littermate controls. Numbers adjacent to outlined areas in E indicate percent cells in each gate. DN DCs are defined as CD4-CD8-CD11c+MHCII+ cells. Each dot in F–I represents an individual mouse and error bars indicate mean ± SE of samples combined from three to five independent experiments. Lymph node migratory DCs are defined as MHCIIhiCD11cint and resident DCs, including the CD8+ and 33D1+ DCs, as MHCIIintCD11chi. *p<0.05, **p<0.01, ***p<0.001, Student’s T-test.