Pe labeled anti il 10

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).

We cultured the transfected cells under Th1 and Th2 polarizing conditions. Cell Stimulation Cocktail and Protein Transport Inhibitor (Invitrogen) were added into transfected cells in the last 6 h of incubation. For cell surface staining, we first used 0.1% BSA, 0.05% sodium azide PBS buffer to wash the collected cells. Then, the surface antibody APC-labeled anti-CD4 and PerCP-cy-5.5-labeled anti-CD3 (BD Biosciences) were used to stain the cells in the dark for 30 min at 4 °C. Subsequently, 4% paraformaldehyde was used to fix the cells under room temperature for 8 min in the dark, and 0.1% BSA, 0.05% sodium azide, and 0.1% saponin (Sigma) PBS buffer was used to permeabilize the cells at room temperature for 2 h in dark. After washing, PE-labeled anti-IFN-γ, PE-labeled anti-IL-2, PE-labeled anti-IL-4, PE-labeled anti-IL-10 (BD Biosciences) were respectively used to stain the cells in the dark for 30 min at 4 °C. Stained cells were assayed with flow cytometry (BD Biosciences) and the program FlowJo version 10.0 (Tree Star Inc. USA) was used to analyze the data.

Cell surface phenotyping was performed by flow cytometry, as previously described [32 (link)]. Allophycocyanin (APC)-conjugated anti-CD3 mAbs (BD Biosciences, San Jose, CA, USA) and an equal amount of the mouse IgG isotype control were used in parallel. An analysis of the cytokine production at a single-cell level was performed. Briefly, the treated cells were either fixed with 4% paraformaldehyde and permeabilized with a FACS permeabilizing solution (BD Biosciences) for IFN-γ, TNF-α, IL-4 and IL-10 detection or were fixed and permeabilized with an intracellular fixation and permeabilization buffer (eBioscience, San Diego, CA, USA) for IL-17 detection. The following cytokine-specific mAbs were used: fluorescein isothiocyanate (FITC)-labeled anti-IFN-γ, PE-labeled anti-IL-4, PE-labeled anti-IL-10 (all from BD Biosciences), PE-labeled anti-TNF-α and FITC-labeled anti-IL-17A (eBioscience). Appropriate isotypic negative controls were run in parallel. To determine the frequency of the T cells, the total lymphocytes were first gated by a forward and side scatter and then additionally gated for the CD3 molecule expression. The acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences) and at least 50,000 events per sample were run. The data were analyzed using Cell Quest Pro software (BD Biosciences).