Anti cd86 pacific blue

Manufactured by BioLegend

Sourced in Germany, United States

Anti-CD86-Pacific Blue is a monoclonal antibody that binds to the CD86 surface antigen. CD86 is a costimulatory molecule expressed on antigen-presenting cells and plays a role in T cell activation. This antibody is conjugated to the Pacific Blue fluorochrome, which can be detected using flow cytometry.

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Anti-CD86 (61 citations) CD86 Pacific blue (2 citations)

4 protocols using anti cd86 pacific blue

Dendritic Cell Maturation and T-Cell Activation

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To determine maturation status, DCs were harvested two days after addition of peptides and maturation factor LPS. Cells were stained in FACS buffer (PBS (GIBCO) containing 0.5% BSA (Sigma) and 0.5 mM EDTA (ICN Biomedicals)) for 30 minutes at 4°C with either one of two panels that contained the following maturation markers: anti-CD80-FITC, anti-CD14-PE, anti-DC-SIGN-APC, anti-HLA-DR-Pacific Blue and Live/dead-AmCyan (Invitrogen) (panel 1) or anti-CD83-FITC, anti-CD40-PE, (BD Biosciences), anti-PD-L1-APC (eBioscience), anti-CD86-Pacific Blue (BioLegend) (panel 2). Live/dead-AmCyan (Invitrogen) was included in both panels. For analysis of the co-culture, the following markers were used: anti-CD8-FITC (Sanquin), anti-CD3-PerCP, anti-TNFα-PE-Cy7, anti-IFN-γ-APC (BD Biosciences), anti-CD4-Pacific Blue (eBioscience) and Live/dead-AmCyan (Invitrogen). Four hours prior to staining, Brefeldin A (BD Biosciences) was added to the culture; then cells were stained using the Cytofix/Cytoperm kit from BD Biosciences according to manufacturer’s recommendations. Cells were measured using a FACS Canto II (BD Biosciences) and results were analyzed using FlowJo version 9.7.5 software. First, lymphocytes were gated, followed by gating of live cells, then CD3⁺ cells and finally CD8⁺ or CD4⁺ cells were placed in a quadrant with TNF-α⁺ or IFN-γ⁺ cells.

Rosendahl Huber S.K., Luimstra J.J., van Beek J., Hoppes R., Jacobi R.H., Hendriks M., Kapteijn K., Ouwerkerk C., Rodenko B., Ovaa H, & de Jonge J. (2016). Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance. PLoS ONE, 11(6), e0156462.

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Characterizing Dendritic Cell Phenotype

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Before staining, FcγR were blocked using the Human TruStain FcX Solution (BioLegend) for 10 min at room temperature in PBS 2% FBS. Cells were then stained for 15 min at 4°C in PBS 2% FBS with one or more of the following Ab: anti- CD14-AF700 (BioLegend), anti-DC-SIGN-AF647 (BioLegend), anti-CD1a-AF488 (BioLegend), anti-CD83-PE (BioLegend), anti-CD86-Pacific Blue (BioLegend), anti-CD40 PE (BioLegend), anti-CD80 Pacific Blue (BD Biosciences), anti-PD-L1 APC (BioLegend), anti HLA-DR A700 (BioLegend).

Olagnier D., Peri S., Steel C., van Montfoort N., Chiang C., Beljanski V., Slifker M., He Z., Nichols C.N., Lin R., Balachandran S, & Hiscott J. (2014). Cellular Oxidative Stress Response Controls the Antiviral and Apoptotic Programs in Dengue Virus-Infected Dendritic Cells. PLoS Pathogens, 10(12), e1004566.

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Characterization of Immune Cell Markers

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The expression of surface markers was characterized by flow cytometry and the use of specific antibodies. On day 0, 6 or 8, non-adherent cells were harvested and collected by centrifugation. The cells were re-suspended in a solution of selected antibodies (PBS, 2mM EDTA, 0.5% BSA) and incubated at 4 °C for 20 min in the dark. Afterwards, 400 µL of PBS was added and the samples were centrifuged at 300× g at 4 °C for 5 min. The cells were then re-suspended in 400 µL of PBS and analysed on Attune NxT flow cytometer (Life Technologies, Carlsbad, CA, USA). The following monoclonal antibodies were used: anti-CD14 Pacific Orange (ThermoFisher Scientific), anti-DC-SIGN APC/Fire 750, anti-CD80 AlexaFluor 488, anti-CD86 Pacific Blue, anti-HLA-DR PerCP, anti-CD85k APC, anti-CD274 PE (All from Biolegend), anti-CD85k FITC, anti-CD274 APC, anti-CD85d PE (all from Miltenyi Biotec, North Rhine-Westphalia, Germany). For isotype control IgG1, IgG2a and IgG2b cocktail was used (all from Biolegend). The results are expressed as median fluorescence intensity (MFI) values.

Weiss M., Anderluh M, & Gobec M. (2021). Inhibition of O-GlcNAc Transferase Alters the Differentiation and Maturation Process of Human Monocyte Derived Dendritic Cells. Cells, 10(12), 3312.

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Characterization of Dendritic Cell Surface Markers

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The levels of membrane markers were characterized by flow cytometry using fluorescently labelled antibodies. On day 0 and 5, nonadherent cells were harvested and collected by centrifugation. Cells were resuspended in 100 µL of culture media with antibodies, and the cells were incubated at room temperature for 20 min in the dark. Afterward, cells were diluted with 400 µL of PBS and analyzed using an Attune NxT flow cytometer. The following monoclonal antibodies were used: anti-CD14 Pacific Orange (ThermoFisher Scientific, MA, USA), anti-DC-SIGN APC/Fire 750, anti-CD80 AF 488, anti-CD83 APC/Fire 750, anti-CD86 Pacific Blue, anti-HLA-DR PerCP, anti-CD85k APC, anti-CD207a PE (All from Biolegend, San Diego, CA). For the purpose of isotype control, an IgG1, IgG2a and IgG2b cocktail was used (all from Biolegend, San Diego, CA). The concentration of cells in a suspension was 1,000,000 per millilitre. Firstly, 150 µL of suspension of dendritic cells were transferred into a new one-well culture slide. The next step was washing cells with sterile PBS twice and then adding the solution of 1. This volume was 5 µL (for concentration 10 -5 M) and 0.5 µL (for concentration 10 -7 M). Cells were ready for analysis with fluorescent microscope after 15 or 30 min of incubation.

Biagiotti G., Purić E., Urbančič I., Krišelj A., Weiss M., Mravljak J., Gellini C., Lay L., Chiodo F., Anderluh M., Cicchi S, & Richichi B. (2021). Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging. Bioorganic chemistry, 109.

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