Leaf purified anti mouse cd28

Splenic mononuclear cells were isolated from mice by mouse lymphocyte separation medium (Dakewei, Shenzhen, China) as previously described (17 (link)). Single-cell suspensions were separated by mechanical disruption of mouse spleens through 70-μm cell strainers and were harvested by gradient centrifugation at 800 g for 30 min, and washed in PBS twice for the experiment. Purified CD4⁺ T cells were negatively selected using the MojosortTM Mouse CD4⁺ T Cell Isolation kit (Biolegend, San Diego, CA, USA). The purity of CD4⁺ T cell was > 95% (Supplementary Figure S1A), and the cells were cultured in RMPI 1640 medium with recombinant murine IL-2 (20 IU/ml, Novoprotein, Shanghai, China). The cells were stimulated with plate-bound anti-CD3 (2.5 µg/ml, LEAF™ Purified anti-mouse CD3ϵ, Biolegend, San Diego, USA) and soluble anti-CD28 (2.5 µg/ml, LEAF™ Purified anti-mouse CD28, Biolegend, San Diego, USA), and the cells were incubated for 24 h for further experiments.