Anti human foxp3 pe

C5aR expression in human PBMCs and cultured splenocytes were determined with multicolor flow cytometric analysis. Briefly, single cell suspensions were first blocked with Fc Block (BD Pharmingen, San Diego, CA) for 15 minutes and then incubated with antibodies for 20 minutes at room temperature. After being washed with buffer (PBS plus 1% BSA), the cells were analyzed with a FACSCanto II flow cytometer (Becton Dickinson, USA). Foxp3 intracellular staining was performed using an eBioscience kit (Cat#00-5521-00) according to the manufacturer’s protocol. Anti-human CD3 PE, Anti-human CD4 FITC, anti-human CD14 APC, anti-human CD25 APC, anti-human Foxp3 PE, anti-human C5aR PE, anti-mouse CD3 PE, anti-mouse CD4 FITC, anti-mouse CD14 APC, anti-mouse C5aR-APC, anti-mouse CD25 APC, and anti-mouse Foxp3 PE (eBioscience, USA) were used for these studies. The data were analyzed in FlowJo software (TreeStar).

First, cells were rinsed with FACS buffer and then stained for the cell surface proteins CD4 and CD25 for 30 min at 4 °C. The cells were then fixed and permeabilized by using the fixation/permeabilization kit followed by the Foxp3 antibody incubation for one hour. Cells were subsequently washed and resuspended in FACS buffer for flow cytometry analysis. Before subjecting cells to FACS analysis, all the samples were passed through a 0.40 μm filter and stained with SYTOX Orange Dead Cell Stain (invitrogen). Samples were run on a BD FACSCanto II (Becton and Dickinson) and sorted with a FACSArial cell sorter (Becton and Dickinson) using the Diva software package. Analyses were performed with either Diva or FlowJo_v10 software. Graphs were generated with Prism 8 software (GraphPad Software, Inc.). The antibodies used for staining were anti-human/ mouse CD4 FITC, anti-human CD25 APC, and anti-human Foxp3 PE (eBioscience). Proper controls were also used through the sample preparation process, including a negative control and single-color controls for each antibody.

Cell culture medium for PBMCs and DCs was RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS (Gibco, Carlsbad, CA, USA) and 100 U/mL streptomycin/penicillin. Anti-human CD3, anti-human CD28, anti-human CD4-FITC, anti-human CD25-APC, anti-human Foxp3-PE, anti-human IFN-γ-PE, anti-human IL-17-PE, anti-human CD11c-APC/Cy7, anti-human HLA-DR-PerCp/Cy5.5, anti-human CD40-PE, and anti-human CD86-PE were all from eBioscience, San Diego, CA, USA. TRIzol, PrimeScript RT reagent kit, and SYBR Green Master Mix were from Takara Biotechnology, Dalian, China. Recombinant human GM-CSF and recombinant human IL-4 were obtained from PeproTech, Rocky Hill, NJ, USA. Meanwhile, human magnetic CD14 isolation kit and CD4 isolation kit were bought from Miltenyi Biotec, Auburn, CA, USA. Lipopolysaccharides (LPS) were from Sigma-Aldrich, St Louis, MO, USA, and recombinant human IL-37 was bought from AdipoGen AG, Liestal, Switzerland. Oxidised low density lipoprotein (oxLDL) was acquired from Yiyuan, Wuhan, China, and phorbol myristate acetate, ionomycin, and monensin were from Alexis Biochemicals, San Diego, CA, USA.

Cell surface staining with antibodies conjugated with fluorochromes was performed as previously described.^{26 (link)} The following anti-human antibodies conjugated with fluorochromes were purchased from eBiosciences (San Diego, CA): CD14-FITC, CD4-FITC, CD11b-PE, CD25-PE, CD3-PerCP, CD33-PercpCY5.5, HLA-DR-APC, FoxP3-APC, and isotype-matched control antibodies conjugated with fluorochrome. Intracellular staining (ICS) with anti-human FoxP3-PE was performed using the FoxP3 staining buffer set (eBiosciences, San Diego, CA) according to the manufacturer's instructions. As a heterogeneous cell population, human MDSCs could be further divided into 2 subsets, monocytic (M-MDSC, CD14⁺) and granulocytic (G-MDSC, CD14⁻/CD15⁺).^{12 (link),18 (link),20 (link)–23 (link)} Given that G-MDSCs are unavailable in Ficoll-prepared PBMCs, we set the gating strategy for M-MDSCs: CD33⁺CD11b⁺/CD14⁺HLA-DR^Low. Meanwhile, the gating strategy for Tregs was CD3⁺CD4⁺CD25⁺FoxP3⁺. Cells were collected on a FACSCalibur (BD). The data were analyzed using FlowJo software (TreeStar, San Carlos, CA). Appropriate isotype controls were used at the same protein concentration as the test antibodies, and control staining was performed during every FACS.

Cells were stained with anti‐human CD4‐FITC (ebioscience), anti‐human CD25‐APC (ebioscience), anti‐human Foxp3‐PE (ebioscience), and anti‐human CD163‐APC (ebioscience), and analyzed with flow cytometry (Becton, Dickinson and Company).