Recombinant mouse il 2 402 ml

Anti-mouse CD45.1 (A20), anti-mouse CD45.2 (104), anti-mouse CD45 (30-F11), anti-mouse TCR γ/δ (GL3), anti-mouse TCR β (H57-597), anti-mouse CD8α (53-6.7), anti-mouse CD8β (YTS156.7.7), anti-mouse Vγ1.1 (2.22), anti-mouse Vγ1.2 (4B2.9), anti-mouse Vγ7 (F2.67), anti-mouse Ki67 (16 A8), anti-mouse CD25 (PC61), anti-mouse CD44 (IM7), anti-mouse CD45RB (C363-16A), anti-mouse CD103 (2E7), anti-mouse CCR9 (9B1), anti-mouse IFN-γ (XMG1.2), anti-mouse TNF-α (MP6-XT22), anti-mouse IL-17A (TC11-18H10.1), anti-mouse CD4 (GK1.5), and anti-mouse CD326 (G8.8) were purchased from Biolegend. Purified anti-mouse CD3 (145-2C11) was purchased from Bio X Cell. Recombinant mouse IL-2 (402-ML) and human TGF-β1 (240-B) were purchased from R&D Systems. SB431542 was obtained from Selleckchem (Cat# S1067). Cell Proliferation Dye eFluor™ 450 (Cat# 65-0842-90), eBioscience ^TM Foxp3/Transcription Factor Staining Buffer Set (Cat# 00-5523-00) were from eBioscience. Mouse TCR γ/δ T cell Isolation Kit (130-092-125) was from Miltenyi Biotec.

Sorted CD4⁺ T cells were labeled with CFSE (ThermoFisher Scientific) and were cultured in custom ordered Dulbecco’s Modified Eagle Medium (D-MEM, KOHJIN BIO) supplemented with 10% heat inactivated FBS (Hyclone). 2.0 × 10⁵ cells were stimulated in 96-well flat-bottomed plate, which was pre-coated with 2 μg ml⁻¹ anti-CD3e antibody (553068, BD Bioscience) and 2 μg ml^-1 soluble anti-CD28 antibody (553295, BD Bioscience), for two days, and were maintained in D-MEM supplemented with 10 ng ml^-1 recombinant mouse IL-2 (402-ML, R&D system) for 2 to 4 days.