Mhc 2 pe cy7

In order to examine activation status of the cells, BMDCs were treated with Duolac ATP or LPS for 24 h at 37°C. The cells were stained with anti-mouse CD86-FITC, PD-L1-PE, MHC II-PE-cy7, CD11c-APC (all from BD Biosciences) for 20 min at 4°C in the dark. To test the increase of Treg, CTV labeled CD4⁺ T cells cultured with Duolac-treated BMDCs for 3 days were stained with anti-mouse CD4-PE (BD Biosciences). After surface staining, CD4⁺ T cells were fixed and stained with anti-mouse Foxp3-APC mAb (BioLegend, Dedham, MA, United States) using FOXP3 Fix/Perm Buffer Set (BioLegend). In vivo examination, single cells from mLNs and PP were isolated from AD mice. Population changes of DCs and Tregs were examined as aforementioned. To analyze for subpopulation of Th cells, total mLN and PP cells were stimulated with PMA and ionomycin (Sigma-Aldrich) in the presence of brefeldin A for 4 h. After the stimulation, the cells were stained with appropriate combination of anti-mouse CD11c-APC, CD4-bv605, Foxp3-APC, IFN-gamma-PE, IL-4-bv605, and IL-17-APC-cy7 mAb (all from Biolegend). The cells were washed and the expression was examined using a FACSCanto II (BD Biosciences). All flow cytometric data acquired were analyzed with FlowJo software (Tree Star, Ashland, OR, United States).

One percent FITC in 1:1 acetone: dibutyl phthalate was painted on the dorsal side of the ears of WT and KO mice. Ear-draining auricular LNs were collected for analysis 20 and 48 h after the painting. dLNs were digested for 30 min in 100 micrograms/ml DNase I and 0.5 mg/ml Collagenase P at 37°C. EDTA was added for the final 5 min incubation. The single-cell suspensions were stained for flow cytometry: CD45-BV650, CD103-PE, CD11c-PerCP-Cy5.5 or CD11C-VB421, CD11b-eFluor450 or CD11b-APC-Cy7, and MHC II-PE-Cy7 or MHC II- PerCP-eFluor 710 (all from BD Biosciences) for 20 min, washed and fixed with 4% PFA. Thereafter, the cells were washed with 0.5% saponin buffer and stained with Langerin-Alexa Fluor 647 (Dendritics, 929F3.01) for 20 min at RT. The samples were measured with LSR Fortessa flow cytometer (BD) and analyzed with FlowJo software (Treestar). Dendritic cell populations were defined among CD11c+MHCII^hi events as follows: Langerin⁺CD103^- (Langerhans cells), Langerin⁺CD103⁺ (CD103⁺ dermal dendritic cells, DDC), Langerin^-CD11b⁺ (CD11b⁺ DDC) and Langerin^-CD11b^- (double negative DDC).