Extracellular and intracellular staining and flow cytometry

The follows antibodies were purchased from BioLegend (California, USA): phycoerythrin (PE) antimouse PD-L1 (Clone 10F.9G2, Cat# 124308), Allophycocyanin (APC) antimouse CD45 (Clone 30-F11, Cat# 103112), fluorescein-5-isothiocyanate (FITC) or PE antimouse CD3 (Clone 17A2, Cat# 100204; Cat# 100206), FITC or PE antimouse CD4 (Clone GK1.5, Cat# 100406; Cat# 100408), peridinin-chlorophyll-protein-cyanine5.5 (PerCP-Cy5.5) antimouse CD8α (Clone 53–6.7, Cat# 100734), PE antimouse CD49b (Clone HMα2, Cat# 103520), PE antimouse TIGIT (Clone 1G9, Cat# 142104), PE antimouse PD-1 (Clone 29F.1A12, Cat# 135206), PE antimouse TIM-3 (Clone B8.2C12, Cat# 134004), PE antimouse LAG-3 (Clone C9B7W, Cat# 125208), FITC antimouse CD107a (LAMP-1; Clone 1D4B, Cat# 121606), PE antimouse/rat tumor necrosis factor (TNF)-α (Clone MP6-XT22, Cat# 506306), PE antihuman/mouse granzyme B (Clone QA18A28, Cat# 396406), FITC antimouse CD62L (Clone MEL-14, Cat# 104406), PE antimouse/human CD44 (Clone IM7, Cat# 103024) and PE rat IgG2b (κ isotype control; Clone RTK4530, Cat# 400607). FITC antimouse NK1.1 (Clone PK136, Cat# 11-5941-82) was purchased from eBioscience-Thermo Fisher Scientific (USA).

For the preparation of single-cell suspensions of tumor tissues, mice were anesthetized and sacrificed, and the tumor tissues were harvested and placed in a serum-free medium (Cat# H740KJ, Basalmedia) with 0.2% collagenase IV (Cat# C4-22-1G, Sigma-Aldrich, Germany). Then, the tumor tissues were cut into 1–2 mm pieces, digested for 2 hours, and passed through 70 µm nylon filters (Cat# CSS013070, Jetbiofil, Guangzhou, China) to obtain the single-cell suspensions. After that, the collagenase was removed by centrifugation, and the cell pellets were suspended in the serum-free medium and adjusted to 2×10⁷ cells/mL. For the preparation of splenocytes, the spleens were obtained from the sacrificed mice and ground into single-cell suspensions using syringe plungers on 70 µm nylon filters (Cat# CSS013070, Jetbiofil). Then, the cells were counted and adjusted to 5×10⁶ cells/mL. Ascites cells were obtained by paracentesis from the peritoneal cavity of the tumor-bearing mice. After cell counting, the cells were adjusted to 2×10⁷ cells/mL. For tumor cell lines, the adherent cells (4T1, MC38, CT26) were digested with 0.5% trypsin in advance, and the suspension-cultured H22 cells were directly adjusted to 2×10⁶ cells/mL. For extracellular staining, the preprepared single-cell suspensions were incubated with the fluorescent monoclonal antibodies for 15 min at RT. After incubation, the cells were fixed with 4% (w/v) paraformaldehyde (PFA, Cat# G1101, Servicebio, Wuhan, China) solution and directly analyzed using a FACS Calibur cytometer (BD; California, USA). For intracellular staining, the fixed cells were ruptured with 1×permeabilization buffer (Cat# 00-8333-56, eBioscience), and then the corresponding antibodies were added respectively and incubated for 30 min in the dark. After washing with PBS once, an appropriate amount of PBS was added to resuspend the cells and immediately analyzed by flow cytometry. Data analysis was performed using FlowJo software (TreeStar; OR, USA).