Primary Mouse T Cell Activation and Th2 Polarization

Primary CD4⁺ mouse T cells were isolated from C57BL/6J peripheral lymph nodes and spleen using positive selection Dynabeads, according to the manufacturer's instructions (Invitrogen). Cells from three mice were plated at 10⁷ cells per 10 cm dish in three cultures. Cells were stimulated with immobilized biotinylated anti-CD3 (clone 2C11, 0.25 µg/mL, Bio X Cell) and anti-CD28 (clone 37.51, 1 µg/mL, Bio X Cell) bound to Corning 10-cm cell culture dishes coated with Neutravidin (Thermo Fisher Scientific) at 10 µg/mL in PBS for 3 h at 37°C. Cells were left on stimulation for 3 d before being taken off stimulation and split into noncoated dishes in T cell medium supplemented with recombinant human IL2 (20 units/mL). Th2 cells were polarized in medium containing 100 units/mL mouse IL4 and anti-IFNγ (10 µg/mL) as described previously (Steiner et al. 2011 (link)). For restimulation, cells were treated with PMA and Ionomycin (20 nM and 1 µM, respectively) for 4 h before harvest.