Cd68 y1 82a

MDSCs were defined as CD11b⁺/CD33⁺/HLA-DR⁻. Single cell suspensions were surface stained with APC-conjugated anti-human CD11b (ICRF44; BD Biosciences, San Diego, CA), FITC-conjugated anti-human CD33 (HIM3-4; BD Biosciences) and PerCP-Cy5.5-conjugated anti-human HLA-DR (G46-6; BD Biosciences). All samples were prepared according to the manufactures’ instructions. The phenotype of circulating M2-like macrophages was analyzed by intracellular CD68 (Y1/82A; BD Biosciences) and CD163 (GHI/61; BD Biosciences) staining after fixing with 4% paraformaldehyde and permeabilizing by FACS permeabilizing solution (BD PharMingen, San Diego, CA). Flow analysis was performed on a FACS Aria II flow cytometer (BD Biosciences) and data were analyzed using FlowJo 7.6.1 (Tree Star, San Carlos, CA) software.

The stored PBMCs of healthy controls and NSCLC patients were thawed by water with 37°C in water bath pot. Then, 100 μl of cell suspensions was placed in a flow tube and was resuspend by 4 ml of phosphate buffered solution. The cellular mixture was centrifuged for 5 minutes at 1500 rpm and the supernatant was discarded. M2-like macrophages needed to be fixed and broken and then were intracellularly stained with CD68 (Y1/82A; BD Biosciences) and CD163 (GHI/61; BD Biosciences) for 30 minutes at room temperature, while MDSCs were not. MDSCs were surfacely stained with APC-conjugated anti-human CD11b (ICRF44; BD Biosciences, San Diego, CA), FITC-conjugated anti-human CD33 (HIM3-4; BD Biosciences), and PerCP-Cy5.5-conjugated anti-human HLA-DR⁻ (G46-6; BD Biosciences) for 30 minutes at 4°C. Finally, all samples were resuspend by 200 μl of PBS and were analyzed by using flow cytometry (BD Biosciences).