Fjk 16

For ow cytometry analysis, the harvested tumors from each group were minced and incubated for 1 h at 37°C in a digestion buffer comprising 2 mg/ml collagenase D (Roche) and 40 μg/ml DNase I (Roche). Cell suspensions were ltered through a 70 μm cell strainer (Corning) and incubated for 3 min at room temperature in ACK lysis buffer (Gibco) to remove the cell clumps and red blood cells. After washing with FACS buffer (1% FBS in PBS), the cells were ltered through a nylon mesh. Next, the cells were incubated on ice for 30 min in Fixable Viability Dye eFluor TM 450 (Invitrogen) to exclude the dead cells before antibody staining. Then, the cells were washed with FACS buffer and incubated on ice for 30 min in FACS buffer with surface antibodies targeting CD45 (30-F11, Invitrogen), CD3 (17A2 or 145-2C11, Invitrogen), CD8a (53-6.7, Invitrogen), CD4 (RM4-5, Invitrogen), PD-1 (J43, Invitrogen), CD25 (PC61.5, Invitrogen), ICOS (7E.17G9, Invitrogen), CD11b (M1/70, Invitrogen), Ly-6G (RB6-8C5, Invitrogen), F4/80 (BM8, Invitrogen), or Ly-6C (HK1.4, Invitrogen). Cells were further permeabilized using a Foxp3 Staining Buffer kit (Invitrogen) and stained for Foxp3 (FJK-16s, Invitrogen), iNOS (CXNFT, Invitrogen), or Arginase 1 (A1exF5, Invitrogen). The stained cells were analyzed using a CytoFLEX ow cytometer (Beckman Coulter), and the data were analyzed with the FlowJo software (Tree Star Inc.).