Cd86 pe

Rats were euthanized by overanesthesia, disinfected with 75% alcohol on the body surface, and the entire spleen was removed by dissection. The spleen was then washed with ImunoSep cell sorting solution (PBM, China), minced, and placed in a 50 ml test tube with a stainless-steel screen. A single cell suspension was prepared by grinding the spleen with a 5 ml rubber tip syringe while adding ImunoSep cell sorting solution dropwise. Erythrocyte lysis was performed using erythrocyte lysis solution (PBM, China). The cell suspensions were incubated with CD68-PE-Vio770 (Miltenyi, Germany), CD86-PE (Thermo Fisher, USA), and CD163-FITC (Bio-Rad, USA) for 30 min at room temperature followed by flow cytometry (ThermoFisher Attune NxT, USA) for detection. The experimental data were analyzed by Attune NxT software.

To evaluate immune cells in mice brain, brain tissues were cut into pieces and digested with 150 μL collagenase (1 mg/mL) and 250 μL DNase (10 mg/mL) for 1 h at 37 °C. Next, cells were harvested and passed through 70 μm strainer. Red Blood Cell Lysis Solution (Beyotime, China) was used to deplete erythrocytes. To evaluate adherent cells in culture dish, cells were digested by 0.05% trypsin for 3 min and separated as single cell suspension. For surface staining, 1 × 10⁶ cells were blocked with anti-FC receptor antibody at 4 °C for 15 min, then stained with indicated antibodies at 4 °C for 30 min. For intracellular staining, cells were fixed and permeabilized using the intracellular staining kit (ThermoFisher, USA). Flow cytometry was performed on a CytoFLEX (Beckman Coulter, USA) and analyzed using FlowJo software (Treestar, USA). The antibodies used in flow cytometry were: CD86 PE (#12-0861-83), CD206 FITC (#MA5-16870), CD11b APC (#RM2805), Iba1 (#PA5-27436), TNF-α PE-CY7 (#25-7321-82), iNOS APC (#17-5920-82), IL-10 PE (#12-7101-82), Arg1 PE-CY7 (#25-3697-82), CD4 PE (#12-0041-82), CD3 APC (#17-0038-42), CD8 PE (#MA5-17849), CD19 PE (#12-0199-42), CD20 FITC (#11-0209-42), CD56 PE (#12-0567-42), CD25 PE-CY7 (#25-0251-82) and FoxP3 PerCP-CY5.5 (#45-5773-82) were all from ThermoFisher (USA).

After treatment, THP-1 cells were washed and detached using Trypsin (Thermo Fisher Scientific). Subsequently, 1 × 10⁶ cells were resuspended in 100 µL of phosphate-buffered saline (PBS)/2% bovine serum albumin (BSA) solution and incubated with a CD86-PE (5 µL, 12-0869-42, clone: IT2.2, Thermo Fisher Scientific) or CD163-PE (5 µL, 333605, clone: GHI/61, BioLegend, San Diego, CA, USA) antibody for 40 min. After rinse twice, cells were resuspended in 500 µL of PBS/2% BSA solution. The cells were detected Beckman cytoflex flow cytometry (Beckman, USA). The Cytexpert Software was applied for the flow cytometry data analysis. The gating strategy for flow cytometry analysis in Figs. 3 and 6 was provided in Supplementary Figure no. 2.

The phospholipid, cholesterol, distearoyl phosphoethanolamine-PEG2000 (DSPE-PEG2000PDP), DSPC, SPDP crosslinker, FITC-dextran, concanavalin A (ConA), and lipopolysaccharide (LPS) were procured from Sigma Chemical Co. (Saint Louis, Missouri, USA). Purified EUPS with a purity of ≥97% was procured from Tianqi Biotechnology Co., Ltd. (Shanxi, China). A standard high-performance liquid chromatography-refractive index detection method was utilized to purify the polysaccharide preparation (≥97% purity). Rat anti-mouse DEC-205 monoclonal antibody was purchased from Thermo Fisher Scientific Co., Ltd. (Dallas, TX). RPMI-1640, fetal bovine serum, were procured from Life Technologies Corporation (Carlsbad, CA). Cell Counting Kit-8 (CCK-8) was obtained from the Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Fluorescently-labeled anti-mouse monoclonal antibodies (CD80-PE, CD40-FITC, CD86-PE, MHC-II-PE, and CD3-FITC, CD8-APC, CD4-PE) were purchased from eBioscience (San Diego, CA).

Bone marrow-derived dendritic cells (BMDCs) were generated from BALB/CByJ mice as described previously [20 (link)]. At day seven post-isolation, BMDCs were either exposed to 10 µM OR141-killed mesothelioma cells (as described above) at a ratio of 1:1 or LPS (from E. Coli, 0.5 μg/mL). DC maturation was analyzed with antibodies against CD11c-BV421 (BD Biosciences, San Jose, CA, USA, 565452), MHCII (I-A/I-E)-APC (BD Pharm, San Jose, CA, USA, 565367), CD40-PE (BD Pharm, 553791), CD80-PE (eBioscience, San Diego, CA, USA, 12-0801), CD86-PE (eBioscience, 12-0862) or CCR7-PE (BioLegend, San Diego, CA, USA, 120105). Live–dead exclusion was achieved by staining with FVD eFluor780 (eBioscience, 65-0865-14). Flow cytometry analysis was performed on FACS Canto II and data were analyzed using FlowJo software. For mouse vaccination, 2 × 10⁶ DC (in 100 μL PBS) were injected i.p. three times at one-week intervals; in parallel, another group of mice was also injected i.p. with 100 µg anti-CTLA4 (CD152) antibody (Bio X Cell, West Lebanon, NH, USA).

For flow cytometric analysis, cells were blocked with PBS containing 1% bovine serum albumin and 0.1% rat IgG (Sigma-Aldrich, St. Louis, USA) for 30 min. After a washing step, cells were stained with SiglecF-PE or -AlexaFluor647 (Clone: E50-2440, BD Pharmingen, San Diego, USA), F4/80-PerCP-Cy5.5 (Clone: BM8), Ly6G-PE (Clone: 1A8), Ly6C-APC-Cy7 (Clone HK1.4) (all BioLegend, San Diego, USA), and CD11b-FITC or -PE-Cy7 (Clone: M1/7; eBioscience, San Diego, USA). For intracellular staining, cells were incubated with fixation and permeabilization buffer (eBioscience) overnight. Cells were stained with rabbit anti-mouse RELMα (Peprotech, Rocky Hill, USA) followed by donkey anti-rabbit IgG AlexaFluor647 (Clone: Poly4064, BioLegend) and CD86-PE (Clone: GL1) and MHCII-APC (Clone: M5/114.15.2, eBioscience) to determine cell activation. The gating strategy used to identify macrophages, monocytes, eosinophils and neutrophils is shown in Figure 1 (Fig. 1).
IFNγ , IL-10, TGFβ and TNF (all eBioscience) as well as CXCL1/KC and CXCL2/MIP-2 (both R&D, Minneapolis, USA) were measured from peritoneal lavage and serum by ELISA according to the manufacturers’ protocols and analyzed using a plate reader (Molecular Devices) with SoftMax Pro 6.