Pe labeled foxp3 ab

Cells were stained with a fluorescein isothiocyanate-labeled CD4 Ab and a phycoerythrin-cyanin 7 (PE-Cy7)-labeled CD25 Ab (BD Biosciences) and were fixed and permeabilized with a fix/perm solution (eBioscience) according to the manufacturer's instructions. The cells were then incubated with a PE-labeled FOXP3 Ab (BD Biosciences), and those in the control group were stained with a FOXP3 isotype control. Samples were analyzed on a BD FACSCanto II flow cytometer (BD Biosciences), and data were evaluated with FlowJo software (ver. 7.6; TreeStar Inc.).

The spleens of the mice in different groups were aseptically removed 24 h after the last challenge. 10⁶ splenic mononuclear cells were stained with fluorescein isothiocyanate- conjugated CD4 Ab (BD Biosciences, Franklin Lakes, NJ, USA) and were incubated with fix/perm solution (eBioscience, San Diego, CA, USA). Fc receptors on cell surfaces were blocked by treatment with excess mouse Fc block (Pharmingen, USA). After the surface staining, cells were stained with PE-labeled FOXP3 Ab (BD Biosciences, USA) and allophycocyanin-CD25 Ab (eBioscience, USA). Treg cells were analyzed by FACSCanto cytometer (BD Bioscience, USA), and data were evaluated with FACSDiva software (BD Bioscience, USA).