Anti mcd45

Unconjugated anti-CD28 (Clone 37.51), anti-CD3ε (Clone 2C11), anti–mCD8-APC, anti–mCD4-BV786, anti–CD3ε-PE-Cy7, anti-mCD107 (LAMP1)-PE, anti–DX5-FITC, and anti-mCD45 were purchased from BD Biosciences. Anti–H60-PE, anti–mNKG2D-PE, rat IgG2a-PE, and rat IgG2b-PE were purchased from R&D Systems.

Tumor Dissociation Kit (Miltenyi Biotec, #130-096-730) was used for single cell suspensions from mouse tumor tissue. The single cell suspension of spleen was obtained by grinding, and white blood cells were obtained by 5% Ficoll density gradient centrifugation. Red Blood Cell Lysis Solution (10×) (Miltenyi Biotec, #130-094-183) was used to remove the erythrocytes. For cell-surface analysis, cells were stained with Fixable Viability Stain 780 (BD Horizon, #565388), anti-mCD45(BD Pharmingen, #561087), anti-mCD3e (BD Pharmingen, #557984), anti-mCD4 (BD Pharmingen, #561831), anti-mCD8a (BD Pharmingen, #561109), anti-CD11b (BD Pharmingen, #561098), anti-mF4/80 (BD Pharmingen, #743280), anti-mCD86 (BD Pharmingen, #564198) in recommended antibody concentrations and incubated at 4°C for 30 min. For the NK intracellular IFN-γ (BD Pharmingen, #562333) and granzymeB (Miltenyi Biotec, #130-101-356) cytokine staining, cells were fixed and permeabilized after stimulation with Leuko Act Cktl with GolgiPlug (BD Pharmingen, #550583) for 6 h. For the CD206 (BD Pharmingen, #565250) and NK1.1 (BD Pharmingen, #563220) staining, cells were also fixed and permeabilized. Cytofix/Cytoperm Soln Kit (BD Pharmingen, #554714) was used to fix and permeabilize the cells. Flow cytometry data was analyzed using FlowJo version 10.