Mouse primary OT-I T cell transfection

Splenocytes were isolated from OT-I TCR transgenic mice and cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, 100 ng/ml IL-2 and 10 nM OVA_257-264 peptide for 3 days. After that, most cells were OT-I cytotoxic T lymphocytes (CTLs) in the medium. CTLs were then transfected with the PMXs-HA-mCD3ε (YY-FF)-mTFP1 chimera construct by retrovirus. In this construct, the two ITAM tyrosines were mutated to phenylalanines to avoid the interference of tyrosine phosphorylation on CD3 conformation. 7.5 × 10⁵ CTLs (in 1.5 ml RPMI-1640 medium) and 1 ml of retrovirus were mixed in the well of 12-well culture plate. 100 ng/ml IL-2 was added to the system. The plate was spun at 1 200× g, 30 °C for 2 h. After spin infection, the cells were cultured for 10 h. Then we did the spin infection again with new viruses. After 10 h, the cells were cultured with fresh RPMI-1640 medium for 1 day and the transfection efficiency was examined by FACS.