Cd163 pe

Cells were treated according to experimental requirements. After treatment, the cells were washed once with DPBS and the supernatant was carefully aspirated with a vacuum pump. Then pre-warmed 0.25% trypsin was applied to digest cells at 37 °C in an incubator for 1 min. The BV2 microglia medium was added with gentle pipetting, to terminate the digestion, and centrifuged at 1200 rpm at room temperature for 3 min. The supernatant was discarded and the cell pellet resuspended with a rat Fc block (CD16/CD32, 1:100, 101320, Biolegend, San Diego, CA, USA) for 10 min on ice. The cells were labeled with I-A/I-E(MHC Ⅱ)-APC (1:80, 107613, Biolegend) and CD163-PE (1:80, 155307, Biolegend) for 15 min at 4 °C, protected from light. The antibodies were washed out and the cells resuspended with FACS buffer for cytometry analysis with BD LSRFortessa (BD Biosciences). The data were analyzed by designing gates with the Flowjo X 10.0.7 R2 software.

Explanted tumors were immediately transferred to RPMI1640-containing plates on ice and cut into 2–4mm pieces using scalpel. An enzymatic digestion was then performed during 30min at 37°C in medium containing Dispase II (1 mg/ml; Roche), Collagenase I (1mg/ml; Sigma) and DNAse I (0.01%; Roche). Samples were passed through a 70 μm-cell strainer, centrifuged, resuspended in medium and passed through a 40 μm-cell strainer. After one more washing step, cells were resuspended in FACS buffer (PBS containing 3% FCS and 2mM EDTA) and incubated with CD16/CD32 Fc Block (BD) before staining with the following specific antibodies (or their corresponding isotype controls): CD45-APC, CD11b-BV570, MHCII-FITC, CD163-PE, Ly6G-PerCP-Cy5.5, F4/80-PE-Cy7 and Ly6C-APC-Cy7, for 20min on ice (all antibodies from Biolegend). Finally, cells were washed and resuspended in FACS buffer containing 1 μg/ml DAPI and measured at a FACS Canto II (BD).

The following antibodies were used for analyses of mouse bone marrow-derived mouse macrophages and human xenografts in mice: CD11b PE-Cy7, F4/80 Alexa Fluor 700, CD80 Alexa Fluor 647, CD206 PE (BioLegend, San Diego, CA, USA). Antibodies for analysis of PBMC-derived human macrophages included CD11b Alexa Fluor 647, CD14 APC-Cy7, CD80 PE-Cy7, CD163 PE (BioLegend). DAPI stain (Invitrogen, Eugene OR, USA) was added to exclude dead cells.
Acquisition was performed on an LSRII Fortessa flow cytometer (BD-Biosciences, Franklin Lakes, NJ, USA) and sorting was performed on a FACSAria II (BD-Biosciences). Data analysis was performed using FlowJo Version 9.6.4 (Tree Star, Ashland, OR, USA). Live singlets were gated using FSC-W/FSC-H. Gates were drawn by using Fluorescent Minus One (FMO) control tubes. Macrophages were separated from tumor cells by their expression of CD11b and CD14 for human as well as CD11b and F4/80 for mouse macrophages.