Quantification of Regulatory T-cells and Cytokines

After sacrifice, spleens were removed under sterile conditions and spleen cells were prepared as described [5 (link)]. Spleen cells were stained for CD4⁺ CD25⁺ Foxp3⁺ T-regulatory cells with the mouse regulatory T-cell staining kit (eBioscience, #88-8111), according to the manufacturer's instructions. Absolute numbers of CD4⁺ T-cells and CD4⁺ CD25⁺ Foxp3⁺ T-cells were calculated per spleen.
For cytokine measurements, spleen cells were stimulated with OVA (0.2 μg per well), medium for 72 h. Undiluted spleen cell supernatants were screened for cytokine production using the mouse Th1/Th2/Th17/Th22 13plex FlowCytomix Multiplex kit (eBiosciences, #BMS822FF), following manufacturer's instructions. Acquisition was performed on a FACS Calibur flow cytometer (BD Biosciences) and data were analyzed using the eBioscience FlowCytomix Pro Software.