Splenic DCs (1 × 105) or peritoneal macrophages were fed with either no antigen,1 μM OVA (Invivogen), or 50 pM SIINFEKL in Iscove’s modified DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum (FBS). After 3 hours, cells were washed and 1 × 106 splenocytes from OT-I mice were coincubated. Brefeldin A (BioLegend) was included in some of the cultures. OT-I T cell activation was detected by surface staining with anti-CD69 after 4 hours or by intracellular IFN-γ staining after 6 hours in the presence of Brefeldin A. In some experiments, APCs were pretreated with anti-CD86 (1 μg/ml; GL1, BioLegend) and/or anti-CD80 (1 μg/ml; 16–10A1, BioLegend) before coincubation with T cells.
Anti cd80
Manufactured by BioLegend
Sourced in United States, Germany
Anti-CD80 is a monoclonal antibody that binds to the CD80 cell surface protein. CD80 is a co-stimulatory molecule expressed on antigen-presenting cells that plays a key role in T cell activation. The antibody can be used for the detection and analysis of CD80 expression.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd86, by BioLegend (24 mentions)
Anti cd11c, by BioLegend (11 mentions)
Anti cd11b, by BioLegend (8 mentions)
Anti cd40, by BioLegend (8 mentions)
Anti cd45, by BioLegend (7 mentions)
Anti cd4, by BioLegend (6 mentions)
Anti mhcii, by BioLegend (6 mentions)
Anti cd69, by BioLegend (5 mentions)
Anti cd3, by BioLegend (5 mentions)
Anti cd8, by BioLegend (5 mentions)
Anti cd83, by BioLegend (4 mentions)
Anti cd62l, by BioLegend (4 mentions)
Anti hla dr, by BioLegend (4 mentions)
Anti cd206, by BioLegend (4 mentions)
Facscalibur, by BD (3 mentions)
Anti nk1, by BioLegend (3 mentions)
Anti cd19, by BioLegend (3 mentions)
Anti cd44, by BioLegend (3 mentions)
Cellquest software, by BD (3 mentions)
Anti cd68, by BioLegend (3 mentions)
37 protocols using anti cd80
1
Antigen Presentation to CD8+ T Cells
2
Modulation of DC Surface Markers by 2-HG
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To analyze DC surface markers, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF for seven days. DCs were treated with 100 ng/mL LPS for 24 h in the absence or presence of 10 mM D-2-HG or L-2-HG. Cells were harvested and stained with anti-CD1a & anti-HLA-DR (Beckman Coulter, Krefeld, Germany), anti-CD80 (Biolegend, San Diego, CA, USA), anti-CD83 (eBioscience, San Diego, CA, USA), and anti-CD86 (BD Bioscience, Franklin Lakes, NY, USA) at 4 °C for 30 min. After washing, DCs were resuspended in FACS wash buffer. Flow cytometric measurement was performed using a BD FACS Calibur instrument (BD Bioscience).
For intracellular detection of IL-12 p35 and IL-12 p40, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF. After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStopTM, BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/CytopermTM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls. Cells were analyzed using a BD FACS Calibur instrument (BD Bioscience).
For intracellular detection of IL-12 p35 and IL-12 p40, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF. After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStopTM, BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/CytopermTM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls. Cells were analyzed using a BD FACS Calibur instrument (BD Bioscience).
3
Immunohistochemical Analysis of Liver Tissue
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Frozen sections(7 µm‐thick) were fixed with 4% PFA for 30 min, and then washed by PBS for three times. The samples were permeabilized and blocked by PBS containing 0.3% Triton X‐100 (Sigma) and 3% bovine serum albumin (BSA) (Amresco) for 1 h. The primary antibody was incubated overnight at 4 °C. The following primary antibodies were used: anti‐COL‐1 (Abcam, ab6308), anti‐αSMA (eBioscience, 14‐9760‐82), anti‐F4/80 (Abcam, ab6640), anti‐CD80 (Biolegend, 104 705), anti‐Arg1 (Cell Signaling Technology, 93 668), anti‐CD31 (Abcam, ab9498), anti‐Ki67 (eBioscience, 12‐5698‐82), and anti‐ALB (Abcam, ab207327). The samples were incubated with fluorescent secondary antibody for 1 h at room temperature. The samples were washed by PBS for three times. Collagen was stained by Sirius Red staining (Huayueyang Bio‐Technology, GH6044s) according to manufacturer's instructions. In situ zymography was performed by using in situ zymography kit (Genmed, GMS80095.1) according to manufacturer's instructions. Histological images were taken using a microscope (3DHISTECH Pannoramic SCAN) or a confocal laser scanning microscope (Olympus FV3000). Image analysis was carried out using Imaris 9.6.0 with a consistent setup.
4
Cell Surface Marker Evaluation
5
Flow Cytometry Analysis of DC Markers
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The levels of membrane expressed molecular markers were determined by flow cytometry using fluorescence labelled antibodies. On day 7 (two days after DC differentiation, 48 hours following single or mixed DC cultivation) single and mixed DC cultures containing either immature iDCs or poly I:C-activated mature DCs (mDCs) were harvested and cells collected by centrifugation. Appropriate antibodies (see below) were added to the cells, followed by incubation at room temperature for 15 min in the dark. The cells were then washed twice with DPBS and re-suspended in 2% paraformaldehyde. The following monoclonal antibodies were used: FITC labelled anti-CD1a, anti-CD14, anti-CD80, and R-PE labelled anti-DC-SIGN (all from Biolegend, CA, USA), FITC labelled anti-CD83 (IQ Products, NL), FITC labelled anti-CD86 (DakoCytomation, Denmark), and R-PE labelled anti-HLA-DR (Exalpha Biologicals Inc., MA, USA). FITC-IgG1 and R-PE-IgG2a cocktail was used for isotype control (Biolegend). Results were expressed as mean fluorescence intensity (MFI) values after subtraction of the MFI value obtained with the control antibody.
6
Copper Nanoparticle Synthesis and Characterization
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Copper chloride dihydrate (CuCl2·2H2O), sodium hydroxide, polyvinylpyrrolidone (PVP-K30), sodium sulfide nonahydrate, and hydrazine monohydrate (N2H4·H2O) were purchased from Aladin (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit and collagenase IV were purchased from Biofroxx (Einhausen, Germany). The annexin V-fluoresceine isothiocyanate apoptosis detection kit and calcein-AM/PI assay kit were obtained from Yeason (Shanghai, China). SH-mPEG5000 and SH-PEG2000-NHS were purchased from Ponsure Biotechnology (Shanghai, China). Standard ISRIB (M7425) was purchased from AbMole (Houston, TX, USA). Copper ion detection kit was obtained from MesGen Biotechnology (Shanghai, China). Anti-G3BP, anti-TIA-1, anti-CD206, and anti-CD86 primary antibodies were purchased from Proteintech (Wuhan, China). Fluorescent Molecule-conjugated anti-CD206, anti-CD86, anti-CD80, anti-CD3, anti-CD4, and anti-CD8 were obtained from Biolegend (San Diego, CA, USA). Anti-eIF2α and anti-p-eIF2α were purchased from Cell Signaling Technology (Danvers, MA, USA).
7
Inhibition of MAPK Signaling in Immune Cells
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Fucodian from Fucus vesiculosus, PD98059, SB230580 and SP600125 were purchased from Sigma-Aldrich. Anti-CD3 (HIT3a), anti-CD14 (63D3), anti-CD16 (3G8), anti-CD19 (HIB19), anti-CD20 (2H7), anti-CD34 (561), and anti-CD56 (5.1H11), anti-CD80, anti-CD83, anti-CD86, MHC class I and MHC class II were procured from BioLegend (San Diego, CA, USA).
8
Dendritic Cell Activation Protocol
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BMDCs were obtained from the tibias and femurs. Cells were cultured in RPMI 1640 supplemented with 10% FBS, 1 mM Na pyruvate, 10 mM HEPES buffer, 1% L-glutamine, 1% nonessential amino acids, 100 units/ml pen/strep (Gibco-Thermo Fisher Scientific, MA USA) and 50 μM 2-mercaptoethanol (Sigma-Aldrich, Darmstadt, Germany). Cells were stimulated with 20 ng/ml IL4 and 20 ng/ml GM-CSF (cat. 130-097-757 and 130-095-746, Miltenyi, Bergisch Gladbach, Germany) and maintained at 37°C in a CO2 incubator for 5 days. Immature dendritic cells were transfected with 1 µg/ml cGAS agonist (G3-ended Y-form Short DNA, cat. tlrl-ydna) by LyoVac (cat. lyec-12) reagent and activated by adding 1 µg/ml RIG-1 agonist (5’ppp-dsRNA, cat. tlrl-3prna), 10 μg/ml DMXAA (5,6-dimethylxanthenone-4-acetic acid or STING ligand, cat. tlrl-dmx), and 1 μg/ml TLR7 agonist (Gardiquimod™, cat. tlrl-gdq-5) (Invivogen, San Diego, USA) for 24 hours. Mature BMDCs were stained with the following antibodies: anti-PDCA, anti-CD80, anti-I-Ab, anti-CD11b, and anti-CD11c (BioLegend) and analyzed by flow cytometry (BD Biosciences).
9
Stimulation of Dendritic Cell Maturation
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Example 17
This Example shows that ILT4 antibodies 21D9.e.IgG1.3 (ILT4.8 in
Monocytes isolated from cynomolgus monkey PBMC (Non-human Primate CD14 Microbeads, Miltenyi) were cultured in the presence of 62.5 U/mL recombinant human GM-CSF (Peprotech) and 125 U/mL recombinant human IL-4 (Peprotech) for 5 days. Anti-ILT4 antibodies were added to the culture @ 10 ug/mL at the time the culture was set up. Cells were stained for their surface expression level of CD86, CD80, and CD83 using anti-CD86 (clone #IT2.2, BioLegend), anti-CD80 (clone #L307), and anti-CD83 (clone #HIB15e). The samples were acquired on a Cytoflex® cytometer (Beckmen Coulter) and analyzed with FlowJo™ software (Tree Star, Inc, Ashland, Oreg.).
The results, which are shown in
10
Hypertensive Kidney T Cell and DC Profiling
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