Mouse cd4 cd62l t cell isolation kit 2

Naive CD4⁺ T cells were purified by mouse CD4⁺CD62L⁺ T Cell Isolation Kit II (Miltenyi Biotec). Purified naive T cells were stimulated with plate-bound goat anti-hamster antibodies, soluble anti-CD3 (0.25 μg ml⁻¹,145-2C11; Biolegend) and anti-CD28 (0.5 μg ml⁻¹, 37.51; Biolegend) for Th0-non-polarized condition culture. In addition to Th0-non-polarized condition, following stimulation was used for each polarized condition: IL-12 (20 ng ml⁻¹; R&D Systems) and anti-IL-4 (10 μg ml⁻¹, C17.8; Biolegend) for Th1; IL-4 (100 ng ml⁻¹; R&D Systems), anti-IL-12 (10 μg ml⁻¹; Biolegend) and anti-IFNγ (10 μg ml⁻¹, XMG1.2; Biolegend) for Th2; IL-6 (3 ng ml⁻¹ or indicated concentration; R&D Systems), TGF-β1 (0.3 ng ml⁻¹; R&D Systems), anti-IL-4 (10 μg ml⁻¹, C17.8; Biolegend) and anti-IFNγ (10 μg ml⁻¹; XMG1.2; Biolegend) for Th17; and IL-2 (20 ng ml⁻¹; R&D Systems), TGF-β1 (3 ng ml⁻¹; R&D Systems), anti-IL-4 (10 μg ml⁻¹, C17.8; Biolegend) and anti-IFNγ (10 μg ml⁻¹, XMG1.2; Biolegend) for Tregs. For signal transduction studies, STA21 (STAT3 inhibitor; Santa Cruz Biotechnology) and SMAD3 inhibitor (SIS3; Santa Cruz Biotechnology) were added to cultures on day 0.

Naïve CD4⁺ CD62L⁺ T cells from BALB/c mice were isolated from fresh spleens using a mouse CD4⁺ CD62L⁺ T Cell Isolation Kit II (Miltenyi) with a MiniMacs Separator in combination with LS and MS columns according to the manufacturer’s instructions. The purity of the cells after separation was routinely > 85% as determined by flow cytometric analysis. For in vitro polarization of Th17 cells, purified naïve T cells were incubated in 24-well plates precoated with anti-CD3 (5 μg/ml), anti-CD28 (1 μg/ml), IL-2 (5 ng/ml), anti-IL-4 antibody (2 μg/ml), anti-IFN-γ antibody (2 μg/ml), transforming growth factor-β (TGF-β, 5 ng/ml), interleukin-6 (IL-6, 20 ng/ml), and IL-23 (10 ng/ml) (all from R&D Systems) with or without EgPSC-ESPs (10 μg/ml) [24 ]. After 72 h of culture, cells and supernatants were collected for further analysis.