Pe mouse anti human cd163

Macrophages and cells extracted from the tumors of nude mice were made into single-cell suspensions. The suspensions were mixed with antigen specific fluorescent antibodies and incubated on ice for 2 h (PE Mouse anti-Human CD163, APC Mouse anti-Human CD86, FITC Mouse anti-Human CD206, PerCPMouse anti-Human CD80, all from Biolegend, USA). Cells were washed twice by adding 3 ml of the flow buffer and were then resuspended in 600 μl of the flow buffer. Flow cytometry was performed using the FACSCalibur flow cytometer (BD Biosciences, USA). Flow cytometry results were analyzed using the FlowJo software (FlowJo, USA).

To evaluate the expression of surface proteins on macrophages including CD206 and CD163, polarized and nonpolarized macrophages were collected and resuspended in cold PBS containing 2% BSA (Sigma, MO, USA), then incubated with FITC mouse anti-human CD206 (BD Biosciences, CA, USA) and PE mouse anti-human CD163 (BioLegend, CA, USA) on ice for 30 min. To further confirm the stemness of SCCHN cells, Tu686 cells were dissociated into single-cell suspension and resuspended in cold PBS containing 2% BSA (Sigma, MO, USA), then stained with PE-Cy5 anti-human CD133 antibody (BioLegend, CA, USA) and FITC mouse anti-human CD44 antibody (BioLegend, CA, USA) on ice for 1 h. Cells were analyzed by flow cytometry with a FACS Calibur instrument (BD Biosciences, CA, USA). All data was analyzed using FlowJo software (Tree Star Inc., OR, USA). The experiments were performed in twice.

We treated macrophages to prepare a single cell suspension and incubated with antibodies (PE Mouse anti-Human CD163, FITC Mouse anti-human CD11b, PC5.5 Mouse anti-human 7AAD, all from BioLegend, USA) at 4°C for 1 h. Next, we used flow buffer to wash cells twice, then centrifuged and resuspended them in 0.1 mL of the flow cytometry buffer for the analysis. We performed flow cytometry (Cytoflex, USA) and analyzed the results using Cytoflex software.
For the mouse model, we used collagenase digestion to obtain single-cell suspensions. The cells were then stained with fluorescence-labeled antibodies against F4/80, CD11b, and CD163 (BioLegend, USA).