Anti pd 1 29f 1a12

Draining lymph nodes (dLNs) were harvested and single-cell suspensions were prepared. For the exclusion of dead cells, the Zombie fixable viability kit (BioLegend) was used. Cells were incubated in FACS staining buffer (PBS containing 1% BSA and 5 mM EDTA) with anti-FcγIII/II receptor antibody (BD), and anti-CD45 (30-F11, BioLegend), anti-CD4 (Gk1.5, BioLegend), anti-CD8 (53–6.7, BioLegend), anti-CD3e (145-2C11, BioLegend), anti-B220 (RA3-6B2, eBioscience), and anti-PD-1 (29F.1A12, BioLegend) antibodies. For intracellular IFN-γ and Gzm B staining, cells were stimulated with 25 ng/ml PMA and 1 µg/ml Ionomycin in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete RPMI) with monensin (Golgi Stop, BD). After five hours of incubation, cell surface staining was followed by intracellular cytokine staining using the Fix/Perm Kit (BD) in accordance with the manufacturer’s instructions with anti–IFN-γ (XMG1.2, BD) and anti-Gzm B (NGZB, eBioscience) antibodies. Fluorescence-minus-one controls were used as negative controls. Cells were acquired on the Gallios (Beckman-Coulter) and data were analyzed using the FlowJo software (v7.6.5).

Tissues were excised from mice, minced and digested in Collagenase for 30 min. The samples were filtered through a 40-μm filter. Erythrocytes were removed with Lysing buffer (BD Biosciences). The samples were then incubated for 10 min with anti-CD16/CD32 blocking antibodies (BD Biosciences). The cells were stained with the following antibodies: anti-CD206 (C068C2, BioLegend), anti-CD11c (HL3, BD Biosciences), anti-CD11b (M1/70, BioLegend), anti-CD45 (30-F11, eBioscience), anti-F4/80 (CI: A3-1, BD Bioscience), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD4 (GK1.5, BioLegend), anti-TCRβ (H57-597, BioLegend), anti-PD-1 (29F.1A12, BioLegend), and anti-CD44 (IM7, BioLegend). The samples were washed, incubated with 7-amino-actinomycin D (BD Biosciences) and then analysed on a FACSAria II (BD Biosciences).