The rat anti-mouse CD25 mAb PC 61.5.3 was purified from hybridoma culture supernatants by protein G chromatography. Control rat IgG was purchased from Sigma. For sensitization to DNFB or FITC, mice were painted with the hapten on the shaved abdomen and footpads as previously described 10, 11. To test the effects of CD25 blockade on hapten-presenting DC, mice were treated with i.p. injections of 250 μg of anti-CD25 mAb given on days −1, 0 and +1 of sensitization. To induce CHS responses to DNFB by adoptive transfer of hapten-presenting DC, mice were sensitized with DNFB and DC were purified from cells suspensions of skin-draining
LN harvested on day +2 post-sensitization using anti-CD11c mAb-coated microbeads (Miltenyi Biotec, Auburn, GSK3235025 mouse CA). The purity of DC was always ≥80%
as assessed by flow cytometry and 4×105 DC were injected intradermally into the lower abdominal area of each animal. On day +5 DC-transferred and, as a negative control, non-transferred mice were challenged with 10 μL of 0.2% DNFB on both sides of each ear. Ear thickness was measured in a blinded manner at 24 h intervals after challenge as previously described 10. The magnitude of ear swelling responses is presented as the mean increase of each group of three mice (i.e. six ears) ±SEM over the thickness measured just prior to hapten challenge on day +5 post-transfer. ELISPOT assays to enumerate hapten-specific T cells producing IFN-γ were performed as
previously described 11, 13. selleck chemicals Skin draining LN cell (LNC) suspensions were prepared from FITC-sensitized mice on day +2 post-sensitization. Two-color flow cytometry analyses were performed as previously described 30. To specifically detect hapten-bearing LC, LNC were obtained at 72 h after sensitization with FITC and were fixed, permeabilized and stained with AlexaFluor 647-labeled anti-CD207 mAb. CD11c+FITC+ or CD207+FITC+ cells were gated and their percentage Dapagliflozin in the total LNC population was evaluated for each analyzed sample. Total numbers of LC in the skin-draining LN of each mouse were calculated based on the percentage of LC in analyzed cell aliquot. To evaluate apoptosis of DC in vitro, LN were pooled from five to ten FITC-sensitized mice at 24 h post-sensitization, and DC were purified from LNC suspensions using anti-CD11c mAb-coated microbeads. Then, 105 DC aliquots were cultured with 2×105 cell aliquots of purified CD4+CD25+ or CD4+CD25− T cells for 4 or 16 h. The cells were then stained with APC-labeled anti-CD11c mAb, washed and incubated with Annexin-V-PE for 10 min at RT. The data were analyzed using CellQuest and FlowJo software. DC were purified from pooled LNC of sensitized WT or lpr mice as described above.