Anti fcr

Cultured cells were stained using mAb specific for mouse cell surface markers conjugated to FITC, PE, PE-Cy5, PerCP, PE-Cy7 (BD Biosciences Pharmingen; eBioscience, San Diego CA; Serotec LTD, Oxford, UK; BioLegend, San Diego CA; and Immunological Sciences, Rome, Italy) at a concentration of 5 μg/ml in 1%BSA-PBS for 20 min at 4°C. Before staining with specific mAb, the Fc receptors were blocked using anti-FcR (BioLegend) at a concentration of 5 μg/ml for 10 min at 4°C. The following mAbs were used: CD4 (GK1.5; RM4-5) CD8 (53–6.7), CD3 (17A2; 145-2c11), CD69 (H1.2F3), MHC class II (14-4-45), CD279 (PD-1; 29F.1A12), CD274 (PD-L1; 10F.9G2), CD273 (PD-L2; TY25) and B220 (RA3-6B2). Single cell events were gated by forward scatter area versus height and side scatter for size and granularity. Cells were analyzed using a FACSCalibur flow cytometer (Beckton Dickinson Immunocytometry System, San Jose, CA) and Cell Quest analysis software. Staining of samples with isotype controls was used as a reference to determine positive and negative populations.

Tumor tissues were digested by 1 mg/mL collagenase type IV (Sigma, USA) and 0.2 mg/mL DNase type I (Sigma, USA) for 30 min at 37°C. Single‐cell suspensions of the spleen, lymph nodes, and tumor were obtained. Cells were blocked with anti‐FcR (Biolegend, USA) and then stained with antibodies against CD3, CD8, CD4, CD11b, Ly6G, and Ly6C (Biolegend, USA). All the samples were collected on a FACS Calibur Flow Cytometer (BD, USA). The data were analyzed by using FlowJo software (Tree Star Inc., USA).