Anti cd11c pe cy5

Mouse lung tissue was digested with collagenase to generate a single-cell suspension. Lung cells or cultured cells were stained with CD11c (anti-CD11c-PE-Cy5.5, e Bioscience, USA), MHCII (anti-MHCII-PE, BD Pharmingen, USA) and GITRL (anti-GITRL-BV421, BD Pharmingen, USA) to identify CD11c⁺MHCII⁺GITRL⁺ DCs. For the analysis of Tregs, lung cells or cultured cells were first stained for CD4 (anti-CD4-APC-Cy7, BD Pharmingen, USA) and CD25 (anti-CD25-PE, BD Pharmingen, USA) and then fixed and permeabilized for intracellular Foxp3 (anti-Foxp3-APC, e Bioscience, USA) staining. For detecting Th1, Th2 and Th17 cells, lung cells or cultured cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μg/ml) for 6 h in the presence of Golgi Stop solution. After stimulation, cells were incubated with surface markers for CD4 (anti-CD4-FITC, BD Pharmingen, USA), permeabilized and stained for the intracellular cytokines IL-4 (anti-IL-4-APC, BD Pharmingen, USA), IFN-γ (anti-IFN-γ-BV421, BD Pharmingen, USA) and IL-17 (anti-IL-17-PE, BD Pharmingen, USA). Data were acquired using FACS Canto II (BD Biosciences, USA) and analyzed with FlowJo software.

DC activation in the mandibular lymph node, inguinal lymph nodes and popliteal lymph nodes was determined at 24 h and 48 h after vaccination using the various vaccine formulations, as described in Section 2.8. The lymph nodes were harvested and prepared as single cell suspensions. The cells were then washed, blocked, and stained using anti-CD11c-PE-Cy5.5, anti-MHC II-APC, anti-CD80-FITC, and anti-CD86-PE (eBioscience). Finally, flow cytometry was performed on a BD FACSCalibur flow cytometer.