Anti cd28 antibodies clone 37

For activated CD8⁺ T cells, T-25 or T-75 flasks were coated with 5 µg/mL anti-CD3ϵ antibodies (clone 145-2C11; 100340, BioLegend) in phosphate-buffered saline (PBS) for 16–24 hr at 4°C before seeding CD8⁺ T cells. Immediately after magnetic isolation, CD8⁺ T cells were resuspended in complete medium (RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 µM 2-mercaptoethanol and 10 mM HEPES) and seeded in the antibody-coated flask at 3–5×10⁵ cells/cm² together with soluble 1 µg/mL anti-CD28 antibodies (clone 37.51; 102116, BioLegend). Cells were cultured for 48 hr at 37°C in a humidified 5% CO₂ atmosphere. For naïve CD8⁺ T cells, magnetically isolated T cells were resuspended in the complete medium and were seeded in a 10-cm petri dish or T-75 flask at 1–2×10⁶ cells/cm² and cultured in the presence of 20 ng/mL recombinant mouse interleukin-7 (rmIL-7; 402-ML-020/CF, R&D Systems, Minneapolis, MN) for 24 hr at 37°C in a humidified 5% CO₂ atmosphere. In some experiments, up to 1×10⁷ naïve CD8⁺ T cells were labelled with 5 µM CellTrace™ Violet (C34571, Thermo Fisher) for 20 min at 37°C in 1 mL complete medium before culturing them with rmIL-7 prior to nucleofection.