Mouse intracellular cytokine staining starter kit

Single-cell suspensions (1 × 10⁶ cells/ml RPMI 1640 medium containing 10% FBS) prepared from the inguinal and axillary LNs of mice sensitized with DNFB for 5 days were cultured in the presence or absence of 100 µg/ml DNBS for 3 days. The relative cell numbers were determined as the relative fluorescence intensity using a Cell Counting Kit-F (Dojindo). The amounts of IFN-γ and IL-17 in the culture supernatants were measured using Quantikine ELISAs according to the manufacturer's instructions (R&D Systems). For the reconstitution assay, CD4⁺ or CD8⁺ T cells (1 × 10⁶/well) and CD11c⁺ DCs (1 × 10⁵/well) purified from DNFB-sensitized mice using the MACS system (Miltenyi Biotec; purity: >95% and 70%, respectively) were co-cultured in the presence of 100 µg/ml DNBS for 24 hr, and the amounts of IFN-γ or IL-17 in the culture supernatants were measured. For the measurement of the proportion of IFN-γ- or IL-17-producing cells among all LN cells, the cells were stained with FITC-conjugated IFN-γ and PE-conjugated IL-17 antibodies using a Mouse Intracellular Cytokine Staining Starter Kit according to the manufacturer's instructions (BD Biosciences) and were analyzed by flow cytometry.