Anti cd206

Human CD14-positive peripheral blood mononuclear cells (PBMCs) (Lonza, Köln, Germany) were cultured in AIM-V medium (Life Technologies, Grand Island, NY, USA) in the presence of granulocyte macrophage colony-stimulating factor for Type 1 (M1) and macrophage colony-stimulating factor for Type 2 (M2) macrophages (CellVivo Human M1 and M2 Macrophage Differentiation Kit, R&D, Minneapolis, MN, USA) for 6 d. Then, the cells were stimulated with 1 μg/mL lipopolysaccharide for 24 h to generate activated M1 and M2 cells. The culture supernatants were collected, and levels of sPD-L1 were measured by ELISA as described above. The expression levels of specific markers for each type of macrophage were evaluated by flow-cytometry using a BD FACS Calibur, and the data were analyzed using BD CellQuest Pro software (BD Biosciences, San Jose, CA, USA). Antibodies used for detection were PE mouse anti-CD80 (clone: L307.4, BD Biosciences) and anti-CD229 (interferon-gamma receptor) (clone: GIR-208, Life Technologies, Carlsbad, CA, USA) for M1, PE mouse anti-CD163 (clone: 215934, R&D) and anti-CD206 (clone: 19.2, BD Biosciences) for M2, and PE mouse anti-PD-L1 antibody (clone: MIH1, BD Biosciences) for PD-L1.

Freshly isolated peritoneal cells or purified peritoneal macrophages and J774A.1 cells treated as described above, were harvested by scraping and stained with anti-F4/80 (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD11b (Bio-Rad Laboratories, Feldkirchen, Germany), anti-MARCO (R&D Systems, Wiesbaden-Nordenstadt, Germany), anti-CD38 (BD Biosciences, San Jose, CA), anti-CD206 (BD Biosciences), or respective isotype controls for 20 min at 4°C. Flow cytometry was conducted on a FACSVerse instrument (BD Biosciences) and analysis was performed using FlowJo software (Ashland, OR).

Cell suspensions were prepared in PBS containing 2% FBS. Cells (1 × 10⁶) were blocked with mouse Fc blocking solution (anti-mouse CD16/CD32 mAb 2.4G2, BD Bioscience) and stained for 30 minutes at 4 °C with the appropriate antibodies. The antibodies used included: human and mouse anti-Foxp3 (1:50, 150D/E4) and anti-Gata3 (1:50, TWAJ) (eBioscience; San Diego, CA, USA); mouse anti-CD3 (1:50, 145-2C11), anti-CD4 (1:50, GK1.5) (BD Bioscience; F4/80), anti-BM8 (eBioscience), and anti-CD206 (1:50, C068C2) (BioLegend); and human anti-CD3 (1:50, SK7), anti-CD206 (1:50, 19.2) (BD Bioscience), anti-CD4 (1:50, OKT4), and anti-CD11b (1:50, ICRF4) (eBioscience). For intracellular staining, a Foxp3/Transcription factor staining buffer set was purchased from eBioscience. Data were acquired using a FACSVerse flow cytometer (BD Bioscience) and analysed using FlowJo software (Tree Star, San Carlos, CA, USA).

Freshly isolated human monocytes and in vitro generated macrophages were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD14, phycoerythrin (PE)-conjugated anti-CD64, anti-CD80, anti-CD206 (BD Biosciences, San Jose, CA, United States) and FITC-conjugated anti-CX3CR1 (BioLegend, San Diego, CA, United States). Monocytes were incubated with antibodies for 30 min at room temperature (RT), then fixed with 4% paraformaldehyde for 15 min at RT, and kept overnight in 4°C prior to analysis. Macrophages were harvested using Macrophage Detachment Solution DXF (PromoCell GmbH, Heidelberg, Germany), stained with antibodies for 30 min at RT and analyzed immediately. Appropriate isotype controls were used as negative controls. Samples were acquired with the BD FACSCanto^TM II system (BD Biosciences). Prior to analysis, monocytes and macrophages were gated based on size forward scatter (FSC) and granularity side scatter (SSC) in order to eliminate other cell types, dead cells, or debris. Analysis was performed using the FlowJo v10.0.7 software (Tree Star, Inc., Ashland, OR, United States). Monocytes were CD14⁺ CD64⁺ CX3CR1⁺ CD206^- CD80^-, whereas macrophages were heterogeneous, with about 50% expressing CD14 and CD64, whereas in general they were positive for CX3CR1 and CD206, and negative for CD80.

To detect CD206 + macrophages, PMA-treated THP-1 cells were first harvested and fixed overnight in 1% paraformaldehyde (PFA) at 4 °C. They were then resuspended in flow cytometry buffer (1 × PBS buffer containing 1% FSA) and stained with anti-CD206 (BD Biosciences, USA) for 30 min at room temperature.