Analyzing Immune Cell Subsets in MLN

Single-cell suspensions from the MLN were prepared and enriched by nylon wool. Referring to previous studies [25 (link),26 ], we calculated the frequency of CD4⁺ CD25⁺FOXP3⁺ Treg cells in individual samples, as well as that of the effector T cells Th1 (CD3⁺CD8^–interferon [IFN]-γ⁺), Th2 (CD3⁺CD8^–IL-4⁺), Th9 (CD3⁺CD8^–IL-9⁺), Th17 (CD3⁺CD8^–IL-17A⁺), and Th22 (CD3⁺CD8^–IL-22⁺IL-17A^–). Cells were stained in PBS containing 1% fetal calf serum with the following antibodies (all from eBioscience): phycoerythrin (PE)-conjugated anti-CD4, fluorescein isothiocyanate (FITC)-conjugated anti-CD25, and PE-Cy5–conjugated anti-FOXP3; PE-conjugated anti-CD3, allophycocyanin-conjugated anti-CD8, FITC-conjugated anti–IFN-γ, anti–IL-4, anti–IL-9, anti–IL-17A, and PerCP-eFluor–conjugated anti–IL-22. Homotype-independent antibody was used as the negative control. IFN-γ, IL-4, IL-9, IL-17A, and IL-22 intracellular staining was performed after 4-h stimulation with 25 ng/mL phorbol 2-myristate 13-acetate, 1 mg/mL ionomycin, and 2 nmol/mL monensin (all from Sigma-Aldrich).