Cd80 pe

Bone marrow-derived dendritic cells (BMDCs) were generated from Balb/CByJ or C57BL/6j mice as described previously [40 (link),61 (link)]. At day 7 post-isolation, BMDCs were either left untreated or exposed to CM treatment (dilution 1:1) for the indicated periods of time. In some experiments, DCs were exposed to 5 µM TGF-beta type I receptor inhibitor SB-431542, 15 µM DGAT1 inhibitor A922500, or 10 µM DGAT2 inhibitor PF-06424439; concentrations were chosen based on our previous work in cancer cells [36 (link)]. DC maturation was obtained by stimulation with 0.3 µg/mL LPS for 18 h and analyzed with antibodies against CD11c-BV421 (BD Pharm, 565452, San Jose, CA, USA), MHCII (I-A/I-E)-APC (BD Pharm, 565367), CD40-PE (BD Pharm, 553791), CD80-PE (eBioscience, 12-0801, San Diego, CA, USA), CD86-PE (eBioscience, 12-0862), or CCR7-PE (BioLegend 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 (version 10.6.2). For DC vaccine generation, Ab1 cells were lysed by triple freeze–thaw processes and added to DCs at day 7. Tumor lysate-loaded DCs were treated on day 8 with 0.3 µg/mL LPS for 8 h before mouse i.p. administration (2 × 10⁶ DCs in 100 μL phosphate-buffered saline (PBS)).

Anti-mouse IL-17A-PE, CD4-APC and IFN-γ-PE-cy7, anti-mouse IgG isotype, anti-human IFN-γ-PE-cy7, CD4-APC and IL-17A-PE and anti-human IgG isotype were obtained from eBioscience. For intracellular expression of IFN-γ and IL-17, CD4+ T cells were incubated for 1 hours with ionomycin (1 μg/mL), PMA (50 ng/mL) and for another 4 hours with brefeldin A (10 μg/ml, Sigma-Aldrich), harvested, washed and fixed before permeabilization.
BMDCs or MD-DCs were stained for 30 minutes at 4 °C with anti-mouse CD86-FITC (eBioscience), CD11c-APC (eBioscience), CD80-PE (eBioscience), CD40-FITC (BioLegend, San Diego, CA, USA), MHCII-PE (eBioscience), anti-human CD86-PE-cy7 (BioLegend), CD40-FITC (BioLegend), HLA-DR-PE-cy5.5 (BioLegend), and CD80-PE (BioLegend).

Dextran sulfate sodium (DSS) and azoxymethane (AOM) were obtained from Yeasen Biotechnology Co., Ltd. (Shanghai, China). Antibodies of p-IκBα, p-p65, IκBα, p65, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States). Human TNF-α and the IL-6 ELISA kit were obtained from eBioscience (Vienna, Austria). Anti–CD16-PE, CD11c-FITC, CD11b-FITC, F4/80-PE, CD-3-APC, CD80-PE, and CD206-PE were obtained from eBioscience (Minneapolis, MN, United States). The Annexin V-FITC/PI apoptosis detection kit was obtained from BD Biosciences (San Jose, CA, United States). Calcein-AM/PI was acquired from Beyotime (Haimen, China). PDTC was purchased from MedchemExpress (Monmouth Junction, NJ, United States). Safflower polysaccharide (SPS) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

BMDC and MØ incubated with or without the peptides (5, 10 or 20 µg/mL) were washed with FACS buffer twice. Then, supernatants were removed and FACS buffer with Live/Dead – BV450 (BD Horizon); MHC-II – PE-Cy7 (clone: M5/114.15.2; Biolegend); F4/80 – FITC (clone: BM8; Biolegend); CD11c – BV711 (clone: N418; Biolegend); CD80 – PE (clone: 16-10A1; eBioscience); CD86 – APC (clone: GK1.5; Biolegend) were added for immunophenotyping and assessment of activation. The cells were incubated for 30 min at 4°C in the dark, washed 2X with FACS buffer and fixed with 1% paraformaldehyde. Sample acquisition was performed on a flow cytometer FACS BD LSRII housed in the Flow Cytometry Core Facility at the Einstein.

BMDCs were isolated from hind limb bones of 6–8 weeks old Balb/c mice (Guangzhou Yancheng Biotech Company, Guangzhou, China). The red blood cells were lysed and the remaining cells were centrifuged at 450 g for 5 min. Then, 2 × 10⁶ BMDCs were seeded to a 6-well plate and cultured with Roswell Park Memorial Institute (RPMI)1640 supplemented with 10% FBS, 20 ng/mL Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF, Sino Biological, Peking, China), and 10 ng/mL Interleukin 4 (IL-4, Sino Biological) and incubated at 37 °C. On day 7, the non-adherent cells were added to the wells of a 24-well plate (1.5 × 10⁶ BMDC/well). DMSO, Compound 2, or Compound 3 (3 μM) were added to the cells and incubated overnight. After 24 h, cells were harvested and supernatants were collected. The cells were washed and stained with fluorophore labeled mAb to measure co-stimulatory molecule expression (CD11c+-FICT, CD86-APC, Biolegend, CD80-PE, eBioscience, Hongkong, China) by flow cytometry. The amount of Interleukin 12p70 (IL-12p70), Interleukin 6 (IL6), interferon gama (INF-γ), and tumor necrosis factor alpha (TNF-α) in the supernatants were quantified by ELISA Kit (Dakewe Biotech Co., Ltd., Guangzhou, China).

Splenocytes were treated with AECs for 48 h, collected in a 1.5‐ml centrifuge tube and then stained with primary antibodies, including anti‐rat CD3‐FITC, CD4‐PE, CD8‐APC, CD45‐RA+, CD161‐PE, CD80‐PE, CD86‐FITC, and CD103‐Alexa Flox@647 (eBiosciences, CA, USA). As reported, we defined the CD3⁺CD4⁺ population as helper T (Th) cells, CD3⁺CD8⁺ population as cytotoxic T (Tc) cells, CD3^‐CD45RA⁺ population as B lymphocytes, CD3^‐CD161⁺ population as natural killer (NK) cells, CD3⁺CD161⁺ population as natural killer T (NKT) cells, and CD80‐PE, CD86‐FITC, and CD103‐Alexa Flox@647 as dendritic (DC) cells (Ayako et al., 2018 (link); Chen et al., 2018 (link); Xu, Wusiman, et al., 2019 (link)). After incubation at 4°C for 30 min, the cells were washed twice and resuspended in PBS before they were transferred to fluorescence‐activated cell sorting (FACS) tubes and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). The cells were gated using forward and side scatter for dead cell exclusion. In each sample, 10,000 events were measured, and data were analyzed using Flow Jo 7.6 software.