Flow cytometry analysis
tumors
TUMORS
Single-cell suspensions of tumors were obtained by enzymatic digestion for 1 h in media containing collagenase A (cat: 11088793001, Roche) and DNAse (cat: 4716728001; Sigma-Aldrich). Fc blocking was performed using CD16/CD32 monoclonal antibody (clone: 93, cat: 14-0161-85; eBioscience) followed by cell surface staining with fluorochrome conjugated antibodies. Dead cells were excluded by staining with LIVE/DEAD Fixable Yellow Dead Cell Stain Kit (cat: L34968; Thermo Fisher Scientific). Intracellular staining was performed after permeabilization using Intracellular Fixation & Permeabilization Buffer Set (cat: 00552100; eBioscience) as per the manufacturer’s instructions.The following antibodies were used: anti-mouse CD45.2 (clone: 104, cat: 560696) and anti-mouse CD11b (clone: M1/70; cat: 557396) were from BD Biosciences, anti-mouse CD3 (clone: 145-2C11; cat: 11-0031-85), anti-mouse CD8a (clone: 53-6.7; cat: 46-0081-82), anti-mouse FoxP3 (clone: FJK-16 s, cat: 17-5773-82), anti-mouse MHC II (clone: M5/114.15.2, cat: 46-5321-82), and anti-mouse CD206 (clone: MR6F3, cat: 12-2061-82) were from eBioscience, and anti-mouse CD4 (clone: GK1.5, cat: 100453) and anti-mouse F4/80 (clone: BM8, cat: 123118) were from BioLegend, respectively. H2Kb SIINFEKL tetramer was a kind gift from Ton N. Schumacher (NKI, Amsterdam, Netherlands) and described previously (