Fitc anti mouse cd8a

FITC anti-mouse CD8a, PE or APC anti-mouse CD4, FITC or Percp-Cy 5.5 anti-mouse CD11b, PE anti-mouse CD11c, PE or APC anti-mouse CD69, PE anti-mouse F4/80, PE anti-mouse Foxp3, FITC or PE anti-mouse IFN-γ, FITC or PE anti-mouse Ly-G and Ly-6C, anti-mouse Gr1, and isotype-matched mAbs were purchased from BD Biosciences; anti-JNK antibody and anti-p-JNK antibody were purchased from Cell Signaling Tecnology. Anti-mouse A20 antibody was purchased from Abcam; Alexa Fluor 488-conjugated donkey-anti-rabbit secondary antibody was purchased from Invitrogen. Fixation/Permeabilization Kit was purchased from eBioscience. OT-I peptide and OT-II peptide were purchased from Invivogen. For cell surface staining, cells were directly stained with either IgG control or fluorescence-conjugated antibodies; for intracellular cytokine staining, cells were permeabilized and fixed after surface staining, and stained with fluorescence-conjugated antibodies or IgG controls. Lymph nodes used in experiments were near the tumors. Analysis was carried out on a FACS calibur flow cytometer (BD Biosciences). CD11b positive cell sorting was performed by Aria I (BD Biosciences). Annexin-V-FLOUS Staining Kit was purchased from Roche and cell apoptosis was determined according to the manufacturer’s protocol.

Splenocytes were harvested, and the single-cell suspension was prepared. PE-CF594 anti-mouse CD3e and FITC anti-mouse CD8a were purchased from BD Biosciences; PE-anti-mouse CD4 antibody, PE-Cy7 anti-mouse CD8a antibody, FITC-anti-CD25 antibody, and PE anti-FoxP3 antibody were purchased from Thermo Fisher. Cells were resuspended in 2% BSA/PBS and stained with fluorochrome-conjugated antibodies at room temperature for 30 min. The cells were washed twice in 2% BSA/PBS and fixed in 1% paraformaldehyde before fluorescence-activated cell sorting (FACS) analysis. All samples were analyzed using the Attune NxT Flow Cytometer (Thermo Fisher). Data analysis was performed using the FlowJo 10 software (Treestar).