Apc labeled anti cd206

TAMs from malignant ascites or pMPHs from peritoneal lavage fluid were stained with FITC-labeled anti-CD14 (Miltenyi Biotech), APC-labeled anti-CD206 (BioLegend), APC-labeled anti-HLA-DR or APC-labeled anti-CD206 (Biozol), PE-labeled anti-CD163, PE-labeled anti-CD64, PE-Cy7-labeled anti-CD16 and APC-labeled anti-CD32 (eBioscience) as described previously [4 (link)]. Intracellular staining was performed with PE-labeled anti-IL-10 (BD Biosciences) after permeabilization for 20 min at 4 C using BD Cytofix Cytoperm Plus Fixation Permeabilization Kit (BD Biosciences). Additionally, APC-labeled anti-CD52 or APC-labeled anti-TIMD4 (Biolegend) was used for surface staining of TAMs and MDMs from healthy donors. Isotype control antibodies were from BD Biosciences, Miltenyi Biotech and eBioscience. Cells were analyzed by flow cytometry and results were calculated as percentage of positive cells and mean fluorescence intensities (MFI).

Gene expression profiles generated from RNA-Seq datasets were verified in TAMs and tumor cells by FACS analysis. Mononuclear cells from patients’ ascites were simultaneously stained with Vioblue-labeled anti-human EpCAM (Miltenyi Biotech) as tumor marker and FITC-labeled anti-CD14 (Miltenyi Biotech), PE-labeled anti-CD163 (eBioscience), or APC-labeled anti-CD206 (Biozol) as TAM marker. In addition, FITC-labeled anti-TGFbeta RIII and PE-labeled anti-LIF-R (all R&D Systems) were used for surface staining. Intracellular staining of permeabilized cells was performed with APC-labeled anti-IL-8 (eBioscience), FITC-labeled anti-S100A8/A9 (Life Technologies) and FITC-labeled anti-S100A14 (antibodies-online) as described previously [7 ]. Isotype control antibodies were purchased from BD Biosciences, Miltenyi Biotech, and eBioscience. Cells were analyzed by flow cytometry and results were calculated as percentage of positive cells and mean fluorescence intensities (MFI).