Cellxvivo mouse th1 cell differentiation kit

Differentiation of CD4⁺ T cells was performed according to our previous study^{17 (link)}. In brief, splenic naïve CD4⁺ T cells were sorted from the indicated mice (8 to 12 weeks of age) by an EasySep™ mouse naïve CD4⁺ T cell isolation kit (STEMCELL Technologies, 19765). Th17, Th1 and Th2 polarizations were then conducted following the instructions of the CellXVivo mouse Th17 cell differentiation kit (R&D Systems, CDK017), the CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and the CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Naïve CD4⁺ T cells were isolated from 8- to 12-week-old mice with an EasySep™ mouse naïve CD4⁺ T cell isolation kit (STEMCELL) according to the manufacturer’s instructions. Sorted naïve CD4⁺ T cells were cultured on irradiated splenocytes (2000 rads) with soluble anti-CD3 (2 µg/ml, 145-2C11; BioXCell) at a ratio of 1:5 in a 24-well plate. The naïve cells were cultured at 1.5 × 10⁶/ml in T cell medium, sodium pyruvate, Hepes, penicillin/streptomycin, gentamicin sulfate, and 2-mercaptoethanol. The following cytokines were added to generate Th17 subset: IL-6 (20 ng/ml; Miltenyi Biotec), TGF-β1 (2 ng/ml; Miltenyi Biotec), anti-IL-4 (10 µg/ml, 11B11, BioXCell), and anti-IFNγ (10 µg/ml, XMG1.2, BioXCell). On day 3 after stimulation, we transferred four 24-wells into one 10-cm dish containing 10 ml of T cell medium, IL-6, TGF-β1, anti-IL-4, and anti-IFNγ. This time we supplemented IL-2 (15 U/ml) into the T cell media and cultured the cells for additional 2 days before analysis. Th1 and Th2 polarizations were performed using CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.