Naive cd4 t cell isolation kit

Human PBMC samples came from the peripheral blood of two male and two female volunteers aged 28–58 years by using lymphocyte separation solution (TBD, Tianjin, China). According to the manufacturer's protocol, the naive CD4⁺ T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada) was used to sort CD4⁺ T cells from freshly isolated human PBMCs. Then, the lymphocyte separation solution (TBD, Tianjin, China) was used to isolate porcine PBMCs from the heparinized peripheral blood of adult landrace pigs. The porcine PBMCs were used as xenogeneic stimulator cells. All experiments in this study were approved by the Medical Ethics Committee and Animal Research Ethics Communities of Sichuan University. All volunteers signed an informed consent form.

Mouse naive splenic CD4⁺ T cells were prepared using the Naive CD4⁺ T Cell Isolation Kit (STEMCELL Technologies). Naive CD4⁺ T cells were activated by plate-bound anti-mouse CD3 (10 μg/mL) and anti-mouse CD28 (1 μg/mL) for 4 days with the following cytokines and antibodies: TGF-β1.2 (2 ng/mL, R&D Systems), IL-6 (40 ng/mL, PeproTech), IL-1β (10 ng/mL, PeproTech), IL-23 (20 ng/mL, PeproTech), anti-IFN-γ (10 μg/mL, BioLegend) and anti-IL-4 (10 μg/mL, BioLegend) for Th17. We cultured cells in 48-well plates with a total volume of 0.5 ml/well of culture medium with 2.5 × 10⁵ cells.

Naive CD4+ T cells were isolated from spleens and lymph nodes of normal mice using the Naive CD4+ T Cell Isolation Kit (STEMCELL, Vancouver, Canada) and cultured with plates pre-coated with anti-mouse CD3/CD28 (both 5 μg/mL, Biolegend, San Diego, CA, USA) for 2–4 h. Cells were incubated in IMDM medium supplemented with 10% FBS (Sciencell, Vancouver, Canada), 2 mM l-glutamine (Gibco, New York, USA), 0.1 mM non-essential amino acids (Gibco, USA), 1% penicillin/streptomycin (Servicebio, Wuhan, China), and 55 μM β-mercaptoethanol (Gibco, New York, USA). For Th17 cell differentiation, 50 ng/mL IL-6, 2.5 ng/mL TGF-β, 5 μg/mL anti-IFN-γ, and 5 μg/mL anti-IL-4 (Biolegend, San Diego, CA, USA) were added to the medium. Acacetin was solubilized with DMSO and added to the medium at beginning of culture in the absence or presence of dimethyl succinate (DES, 5 mM, MedChemExpress, Shanghai, China) and Atpenin A5 (1 μM or 2 μM, MedChemExpress). Fluorescence was detected by flow cytometry on second or 3rd day of the culture.