Anti cd16 32 monoclonal antibody

Multiparameter flow cytometry was performed according to a standard protocol. Single-cell suspensions were incubated with anti-CD16/32 monoclonal antibody (Biolegend, San Diego, CA, United States) for 15 min at room temperature to block Fc receptors before staining with the specific antibodies. Spleen mononuclear cells were then stained with the indicated fluorescent monoclonal antibodies for surface molecules. Intracellular staining was performed using fixation and permeabilization buffers (eBioscience) according to the manufacturers’ instructions.

Tumor samples were minced with scissors before incubation with 66 μg/ml Liberase and 0.2 mg/ml DNase (Roche, Switzerland) in DMEM for 30 min at 37 °C. Single cell suspensions were prepared by filtering through a 100-μm nylon filter strainer and washed thoroughly in Hank’s balanced salt solution (HBSS) buffer supplemented with 2% FBS, 20 mM HEPES, and 5 mM EDTA. Fixable Viability Dye (Thermo Fisher Scientific, USA) was applied to eliminate dead cells in combination with anti-CD16/32 monoclonal antibody (BioLegend, USA) for 15 min on ice in the dark. Then, cells were stained for 30 min in PBS with appropriate dilutions of various combinations of the following fluorochrome-conjugated antibodies: anti-CD206 PE; anti-CD86 APC/Cy7; anti-CD45 APC; anti-CD11b FITC; and anti-F4/80 PE/Cy7; anti-CD4 PE/Cy7; anti-CD3 APC/Cy7; anti-CD25 APC; anti-CD8 FITC; anti-NK1.1 PE; anti-CD45 Brilliant Violet 510; anti-CD45R Brilliant Violet 510; anti-CD335 PerCP/Cy5.5; anti-CD45 Brilliant Violet 421; anti-Ly6G Alexa Fluor® 647; anti-CD11c FITC. For intracellular staining, cells were further permeabilized using a FoxP3 Fixation and Permeabilization Kit (Thermo Fisher Scientific, USA) and stained with anti-FoxP3 PE antibody (Thermo Fisher Scientific, USA). All flow cytometry data was acquired and analyzed as mentioned above.