Cd86 v450

Cells were stained according to the manufacturer’s recommendations with the following antibodies alone or in varying combinations, depending on the experiment: CD14-FITC, CD4-APC and CD45RA-PE (all Immunotools, Friesoythe, Germany), CD40-PE (AbD Serotec, Kidlington, UK), CD86-V450, HLA-DR-PerCPCy5.5, CD1a-PE and CD3-APC/Cy7 (all BD). For surface marker staining, PBS containing 1% FCS and 5mM EDTA (Sigma Aldrich) was used as staining buffer. For intracellular cytokine staining with IFNγ-PerCPCy5.5 (eBioscience), IL17A-PE and IL10-Brilliant Violet 421 (both BioLegend), monensin solution (BioLegend) was added to cell cultures for 6 hours before harvesting. Once harvested, cells were fixed and permeabilized using the fixation and permeabilization buffer set from BioLegend. Staining with FoxP3-Alexa Fluor 647 (BD) was performed with FoxP3 staining buffer set (eBioscience). The samples were analyzed by flow cytometry on a BD FACS Canto II.

Macrophages cultured for 7 days were carefully harvested using Dispase enzyme solution and gentle scraping. Cells were counted and a minimum of 3 × 10⁵ cells were used for each FACS staining. Fc receptors were blocked in 3% BSA in 1× HBSS (Gibco) for 10 min. As this study focused on membrane proteins, permeabilization and fixation of cells were omitted. Instead, cells were re-suspended in PBS and incubated with the respective antibodies for 30 min. Antibodies used for FACS were αCD163-APC and αCD90-PE (both Biolegend), CD11b-V450, CD11c-PE, CD40-FITC, CD45-PE, CD209-Cy5.5, CD80-V450, CD86-V450, CD146-PE, and CD206-FITC (all BD Pharmingen). Unstained cells were used as control, as well as an IgG control. Cells were washed twice after antibody incubation and re-suspended in PBS for counting. Cell sorting was performed on a LSR-II instrument (BD Bioscience) using FACSDiva8 acquisition and analysis software. Cells were gated in three steps; first, cells were discriminated by size employing forward and side scatter (FSC and SSC, respectively). Second, doublets were removed by pulse-geometry gating the area and height of FSC. Lastly, the fluorescence signal of cells positive for respective markers was gated directly, plotting it against the SSC area.

THP-1 cells were treated with 100 ng/mL 2-Acetoxy-1-methoxypropane (PMA; P8139, acquired from Sigma-Aldrich Chemical Company, St Louis, MO) for 24 h to induce differentiation into macrophages, and then with 20 ng/mL IL-4 (AF-200–04-5, Peprotech, Rocky Hill, NJ, USA) for 72 h to induce the differentiation into M2 macrophages. Cell surface antigens including CD11b, F4/80, CD206 and CD86, were tested by flow cytometry. Briefly, M2-polarized macrophages were trypsinized, rinsed with PBS, and resuspended in 100 μL PBS. Next, the cells were probed with antibodies against CD206-APC (550,889, BD Pharmingen, San Diego, CA, USA), CD86-V450 (560,357, BD Pharmingen), CD11b-PCy5.5 (740,861, BD Pharmingen) and F4/80-PE (565,410, BD Pharmingen) for 1 h. The cells were then resuspended with 0.5 mL PBS, filtered through a nylon mesh, and analyzed on a flow cytometer (Becton Dickinson, San Jose, USA).