Ab120499

For cell‐surface analysis, cells were stained with the anti‐mouse Zombie NIR Fixable Viability Kit (423106), and incubated with antibodies against CD45 (103114), CD11b (101205), F4/80 (123121), CD3 (100212), CD4 (100408), and CD8a (100752) at the recommended concentrations at 4 °C for 30 min. For the T‐cell intracellular IFN‐γ (505808) cytokine staining, cells were fixed and permeabilized after stimulation with Phorbol 12‐myristate 13‐acetate (PMA) (ab120297, Abcam, 100 ng mL⁻¹), Monensin sodium salt (ab120499, Abcam, 1 ug mL⁻¹), and Ionomycin calcium salt (5608212, PeproTech, 100 ng mL⁻¹) for 6 h. For the CD206 (141706) staining, cells were also fixed and permeabilized. All flow cytometry antibodies were purchased from Biolegend (San Diego, CA, USA).

To determine immune cell populations, the detached tumors were weighed, cut into pieces and digested with enzymes, including collagenase V (0.32 mg/mL, Biosharp) and hyaluronidase (0.5 mg/mL, Biosharp) for 1 h. After addition of the RBC lysis buffer for 8 minutes at 4 °C, single-cell suspensions were acquired through 200-mesh filters (the single cell suspension of the lymph nodes was directly harvested through the filter). The cells were incubated with FcR blocking reagent (101320) to block non-specific binding sites, and then stained with anti-mouse fluorochrome-labeled Abs against Zombie Violet™ Fixable Viability Kit (423114), Zombie NIR™ Fixable Viability Kit (423106), CD45 (103130), CD3e (100306), CD4 (100422), CD8a (100751), CD11c (117310), CD86 (105012), CD80 (104708). For analysis of T-cell functions, cells were stimulated with Monensin sodium salt (ab120499, Abcam, 1.5 μg/ml), PMA (ab12029, Abcam, 100 ng/ml), and lonomycin calcium salt (5608212, peproTech, 1 μg/ml) for 5 h. Different from surface staining antibodies mentioned above, intracellular IFN-γ (505808) was detected after fixation and permeabilization. All the flow cytometrical detections in this study were carried out on a flow cytometer (Beckman CytoFLEX S). All antibodies (as listed above) were purchased from Biolegend.

For cell-surface analysis, cells were stained with anti-mouse Zombie Violet™ Fixable Viability Kit (423114), Zombie NIR™ Fixable Viability Kit (423106), CD45 (103114), CD11b (101205), CD86 (105012), CD4 (100408), CD8a (100752), Gr-1 (108411), and PD-L1 (124311, 124308) in recommended antibody concentrations and incubated at 4 °C for 30 min. For the T-cell intracellular IFN-γ (505808) cytokine staining, cells were fixed and permeabilized after stimulation with Phorbol 12-myristate 13-acetate (PMA) (ab120297, Abcam, 100 ng/mL), Monensin sodium salt (ab120499, Abcam, 1 ug/mL), and Ionomycin calcium salt (5608212, PeproTech, 100 ng/mL) for 6 h. For the CD206 (141706) and Foxp3 (126408) staining, cells were also fixed and permeabilized. All flow cytometry antibodies were purchased from Biolegend (San Diego, CA, USA).