Pe conjugated anti mhc 2

Tumor masses were minced and digested with a mixture of 0.3 mg/ml DNase I (Sigma-Aldrich) and 0.25 mg/ml Liberase TL (Roche) in serum-free RPMI at 37°C for 25 min. Then the digested pieces were gently pressed between the frosted edges of two sterile glass slides, and the cell suspension was filtered through a 40 μm cell strainer (BD Biosciences). The immune subsets in the tumors were determined by flow cytometry and data were acquired using a FACS flow cytometer (BD Biosciences, San Jose, CA). For intracellular IFN-γ staining, harvested cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 4 h and incubated for the last 1 h with brefeldin A (10 μg/ml). Cells were subjected to intracellular cytokine analysis with anti-IFN-γ antibody. The antibodies used for FACS include APC-conjugated anti-CD45, Pacific blue-conjugated anti-CD8, FITC-conjugated anti-IFN-γ, Alex Flour 488-conjugated anti-p-S6, PerCP/Cy5.5-conjugated anti-CD4, PE-conjugated anti-NK1.1, FITC-conjugated anti-TCRγδ, PE-Cy7-conjugated anti-CD11b, FITC-conjugated anti-Gr-1, and PE-conjugated anti-MHC-II (eBioscience). Data were analyzed by FlowJo software (TreeStar Inc., San Carlos, CA).