Naive t cell isolation kit 2

Naive (CD4⁺CD25⁻CD62L^highCD44^low) T cells from the spleens and lymph nodes of 6- to 9-week-old mice were isolated using a Naive T Cell Isolation Kit II (Miltenyi Biotec). Purified naive CD4⁺ T cells were stimulated with plate-bound anti-CD3/CD28 (2 μg/mL; BD Biosciences). The cells were incubated in the presence of the following cytokines for 3 or 5 days: anti-IL-4 neutralizing antibody (5 μg/mL; BD Biosciences), IL-2 (50 U/mL; PeproTech), and IL-12 (2 ng/mL; PeproTech) for Th1; anti-IFN-γ neutralizing antibody (5 μg/mL; BD Biosciences), IL-4 (30 ng/mL; BD Biosciences), and IL-2 (50 U/mL; PeproTech) for Th2 cells; anti-IL-4/IFN-γ neutralizing antibody (5 μg/mL; BD Biosciences), TGF-β (1 ng/mL; R&D Systems), IL-6 (30 ng/mL; BD Biosciences), IL-1β (20 ng/mL; R&D Systems), and IL-23 (20 ng/mL; R&D Systems) for Th17; IL-2 (100 U/mL; PeproTech) and TGF-β (5 ng/mL; R&D Systems) for iT_reg cells. Th2 cells were harvested at day 5 and were further reactivated by plate-bound anti-CD3/CD28 (2 μg/mL) for 24 h. Cells were incubated with dNP2-VEET (1 μM), dNP2-VIVIT (1 μM), and CsA (20 ng/mL; Calbiochem).

Naive (CD4⁺CD25⁻CD62L^highCD44^low) T cells from the spleens and lymph nodes of 6- to 9-week-old mice were isolated using a Naive T Cell Isolation Kit II (Miltenyi Biotec). Purified naive CD4⁺ T cells were cultured in 96-well plates at 2.5 × 10⁵ per well. Cells were incubated with dTomato, 11R-dTomato, and dNP2-dTomato proteins for 1 h. Following incubation, the cells were harvested and washed with PBS. Intracellular fluorescence was detected by flow cytometry (FACSCanto II; BD Biosciences), and data were analyzed using FlowJo software version 10.0.7 (Tree Star).