In experiment the CD and CD

In experiment 3, the CD4 and CD25 surface molecules were stained using 1:50 of biotinylated anti-CD4 and 1:10 of anti-CD25 antibodies. Subsequently, secondary conjugate, 1:500 of streptavidin-PE and 1:100 goat-anti mouse IgG1-FITC, diluted in FACS buffer were added. The H 89 stained with the isotype control antibody (biotinylated IgG2b antibody and mouse IgG1 antibody) were included. The cells were then fixed and permeabilized with a 100 μl/well of 50% Reagent A (Leucoperm), diluted in FACS buffer, at room temperature in the dark for 30 min. Anti-human Foxp3 mAb diluted 1:20 in Reagent B (Leucoperm) was added. For intracellular cytokine staining system, the cells were stained by addition of 1:50 of anti-swine CD3-FITC mAb. The cells were then fixed and permeabilized. Anti-swine IFN-γ and anti-swine IL-10 mAbs diluted 1:100 in Reagent B (Leucoperm) were added. Subsequently, secondary conjugate, 1:250 of Streptavidin-PETR and 1:100 of goat-anti mouse IgG1-Alexaflur647, diluted in FACS buffer were added. The cells stained with the isotype control antibody (biotinylated IgG1 antibody) were included, and used as the background cut-off. The fluorescent minus one (FMO) staining controls were performed during the establishment and validation of an assay.