Collagenase 1

Fresh tumor and spleen samples were minced, treated with 200 U/ml Collagenase I (Cat#ST2294, Beyotime Biotechnology), and filtered with a 100 μm filter to make a suspension of single cells. 2 × 10^{6 (link)} Cells were stained at 100 μl in PBS. Single-cell suspension is stained with the following antibodies: Live/Dead Fixable Violet Dead Cell (Cat# 2549272, Invitrogen), anti-CD3 (Cat# 100236, Biolegend), anti-CD4 (Cat# 100406; 100408, Biolegend), anti-CD8 (Cat# 100706, Biolegend), anti-Ki67 (Cat# 652425, Biolegend), anti-IFN-γ (Cat# 505860, Biolegend), anti-Perforin (Cat# 154306, Biolegend), anti-Foxp3 (Cat# 126404, Biolegend), anti-CD11b (Cat# 101212, Biolegend), anti-CD11c (Cat# 117352, Biolegend), anti-Gr-1(Cat# 108406, Biolegend), anti-TIM-3(Cat# 119727, Biolegend), anti-LAG-3 (Cat# 125241, Biolegend) and anti-PD-1 (Cat# 135227, Biolegend). Surface antigens were detected by NovoCyte Quanteon (Agilent) after staining with antibodies for 30 minutes in the dark. To detect intracellular cytokines, the samples were fixed with Fixation/Permeabilization Solution KiT (Cat# 554714, BD Biosciences) and incubated with intracellular antibodies for 30 minutes before running the machine. Datas were analyzed by NovoExpress.