Anti cd80 bv421

Manufactured by BioLegend

Anti-CD80-BV421 is a fluorochrome-conjugated antibody that binds to the CD80 cell surface protein. CD80 is a co-stimulatory molecule expressed on antigen-presenting cells that plays a role in T cell activation. The BV421 fluorochrome allows for detection and analysis of CD80-expressing cells.

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8 protocols using anti cd80 bv421

Comprehensive Immunophenotyping of Immune Cells

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Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Reincke M.E., Payne K.J., Harder I., Strohmeier V., Voll R.E., Warnatz K, & Keller B. (2020). The Antigen Presenting Potential of CD21low B Cells. Frontiers in Immunology, 11, 535784.

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Characterization of DRG Myeloid Cells

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DRG single-cell suspensions were blocked for non-specific antibody-binding with anti-CD16/32 Fc-Block (BioLegend 101320, 1:100) and incubated in an antibody cocktail consisting of anti-CD163-PE (BioLegend 156703, 1:200), anti-TLR4-PE/Cy7 (BioLegend 117609, 1:200), anti-CD206-PerCP/Cy5.5 (BioLegend 141715, 1:200), anti-CD11b-APCCy7 (BioLegend 101225, 1:200), anti-CD68-Alexa700 (BioLegend 137025, 1:200), anti-CD80-Bv421 (BioLegend 104725, 1:200), anti-CD86-Bv510 (BioLegend 105039, 1:200), anti-Gr1-Bv605 (BioLegend 10844, 1:200), anti-F4/80-Bv785 (BioLegend 123141, 1:200), and anti-CX3CR1-FITC (BioLegend 149019, 1:200) for 20 min at 4 °C. Exclusion of dead cells was achieved by resuspending cells in FACS buffer containing 10 nM TO-PRO-3 (Invitrogen, Carlsbad, CA, USA, T3605). Flow cytometry from the samples was performed using a BD LSRFortessa device and BD FACSDiva Software (BD Biosciences). Raw data were analyzed using the CytoExplorer R package version 1.1.0 [46 ].

Choconta J.L., Labi V., Dumbraveanu C., Kalpachidou T., Kummer K.K, & Kress M. (2023). Age-related neuroimmune signatures in dorsal root ganglia of a Fabry disease mouse model. Immunity & Ageing : I & A, 20, 22.

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Generating NFAT Reporter T-cell Line

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The Jurkat 76 T-cell line, deficient for both TCRα and TCRβ chains, was kindly provided by Dr. Shao-An Xue (Department of Immunology, University College London). The NFAT reporter cell line (J76-NFATRE-luc) was constructed using lentiviral transfer of pNL[NlucP/NFAT-RE/Hygro] (Promega) into the Jurkat 76 cell line. Single cell clones with the best fold induction were selected based on the induction of luciferase after PMA/Ionomycin stimulation. K562 cell line was obtained from the ATCC and cultured under standard conditions. Artificial antigen presenting cells (aAPC) were constructed using lentiviral transduction of CD80, HLA-DM molecules and different HLA alleles (gBlock ordered from IDT) into K562 cells. The surface expression of CD80 and HLA-DM on K562 was confirmed using anti-CD80-BV421 and anti-HLA-DM-PE antibodies from BioLegend.

Huang H., Wang C., Rubelt F., Scriba T.J, & Davis M.M. (2020). Analyzing the M. tuberculosis immune response by T cell receptor clustering with GLIPH2 and genome-wide antigen screening. Nature biotechnology, 38(10), 1194-1202.

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Comprehensive Immunophenotyping of Myeloid Cell Subsets

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Staining of cell surface markers was performed as described [67 (link)]. The following antibodies were used: anti-CD11b-eF450, anti-MHC II-PE, anti-F4/80-APC-H7 (eBioScience), anti-CD11b-FITC, anti-Ly6G-AF647, anti-Ly6C-Pe-Cy7, anti-CD80-BV421, anti-PD-L1-PE, anti-CD3-PercP-Cy5.5, anti-CD11c-AF647 (Biolegend, London, United Kingdom), anti-Ly6G-PE-CF594, anti-CD8-FITC (Becton Dickinson, Erembodegem, Belgium) and anti-CD45-VioBlue (130-102-775) (Mitenyi Biotec). For intracellular staining, cells were treated with inside FIX and incubated for 20 minutes at room temperature. Cells were incubated with PERM (Miltenyi Biotec) and the anti-arginase-1-PE (R&D systems, Abingdon, United Kingdom) or anti-iNOS-PercP-Cy5.5 (Santa Cruz Biotechnology, Heidelberg, Germany) antibody for 20 minutes at room temperature. Subsequently, cells were washed. Cells stained with isotype matched control antibodies served as a control. Cells were acquired using the LSR Fortessa (Becton Dickinson) and analysis was performed using FlowJo 7.6 (Treestar Inc Oregon, United States of America).

Dufait I., Schwarze J.K., Liechtenstein T., Leonard W., Jiang H., Escors D., De Ridder M, & Breckpot K. (2015). Ex vivo generation of myeloid-derived suppressor cells that model the tumor immunosuppressive environment in colorectal cancer. Oncotarget, 6(14), 12369-12382.

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Generating NFAT Reporter T-cell Line

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Characterization of Tumor Cell Antigen Expression

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Human tumor cell line expression of antigens and receptors was characterized using FACS analysis utilizing the Canto II (Becton Dickinson, Franklin Lakes, USA) platform. Cells were quantified and isolated from culture at 5x10⁵-1x10⁶ cells per FACS tube, stained with anti-CD19 FITC (Miltenyi Biotec Cat#130-113-645; RRID:AB_2726198), anti-CD80 BV421 (BioLegend Cat#305222; RRID:AB_2564407) or anti-CD80 PE (ImmunoTools Cat#21270804; RRID:AB_2923118) and anti-CD86 APC (ImmunoTools Cat#21480866; RRID:AB_2923116), washed twice each before and after antibody application with PBS and incubated at 4°C for 30 min before analysis.
Murine tumor cell line and primary cell expression of antigens was evaluated on the MACSQuant X (Miltenyi Biotec, Bergisch Gladbach, Germany) and Canto II (Becton Dickinson, Franklin Lakes, USA) platforms using anti-mouse CD19 (1D3) APC (ImmunoTools Cat#22270196X2; RRID requested) or anti-mouse CD19 BV510 (6D5) (BioLegend Cat#115545; RRID:AB_2562136), anti-mouse CD80 (16-10A1) PE (BioLegend Cat#104707; RRID:AB_313128) and anti-mouse CD86 (GL-1) PE/Cy7 (BioLegend Cat#105014; RRID:AB_439783) antibodies.

Prinz L.F., Riet T., Neureuther D.F., Lennartz S., Chrobok D., Hübbe H., Uhl G., Riet N., Hofmann P., Hösel M., Simon A.G., Tetenborg L., Segbers P., Shimono J., Gödel P., Balke-Want H., Flümann R., Knittel G., Reinhardt H.C., Scheid C., Büttner R., Chapuy B., Ullrich R.T., Hallek M, & Chmielewski M.M. (2024). An anti-CD19/CTLA-4 switch improves efficacy and selectivity of CAR T cells targeting CD80/86-upregulated DLBCL. Cell Reports Medicine, 5(2), 101421.

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Comprehensive Immune Cell Profiling

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For intracellular cytokine detection, T cells were restimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL, Sigma-Aldrich) in the presence of Brefeldin A (0.2%, BD Biosciences) for 4 h. Cells were then stained with antibodies against T cell markers or their respective isotype control anti-CD25-PECy7 and anti-CD4-FITC (BD Biosciences) for 20 min at 4°C in the dark, fixed, and permeabilized with the Transcription factor buffer set (BD Biosciences) and then stained intracellularly with anti-Foxp3-PE, anti-IFNγ-PE, and anti-IL17A-AF647 antibodies (BD Biosciences) for 30 min at 4°C for flow cytometry detection of Tregs, Th1, and Th17 cells, respectively.
For flow cytometric detection of macrophages, cells were stained with the following antibodies or their respective isotype controls anti-CD40-APCy7 (BioLegend), anti-CD80-BV421 (BioLegend), anti-CD16-V500 (BD Biosciences), anti-CD206-AF488 (BioLegend), anti-CD163-APC (Miltenyi Biotec) for 20 min at 4°C in the dark. All samples were acquired on a MACSQUANT Q10 cytometer (Miltenyi Biotec) and analyzed using FlowJo v7.6.5 software (Tree Star).

Sayegh S., El Atat O., Diallo K., Rauwel B., Degboé Y., Cavaignac E., Constantin A., Cantagrel A., Trak-Smayra V., Alaaeddine N, & Davignon J.L. (2019). Rheumatoid Synovial Fluids Regulate the Immunomodulatory Potential of Adipose-Derived Mesenchymal Stem Cells Through a TNF/NF-κB-Dependent Mechanism. Frontiers in Immunology, 10, 1482.

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Lung Cell Cytokine Profiling and Antibody Neutralization

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Lung cells were rst blocked for Fc receptors with Fc Block (CD16/32, BD Bioscience) and then surfacestained with uorescein-conjugated antibodies in PBS for 30 min at 4 °C under light protection. For intracellular cytokine staining, naïve CD4 + T cells were isolated from the lungs of mice and stimulated with PMA (25 ng/mL, Sigma) and ionomycin (1 μg/mL, Sigma) for 4.5 h at 37 °C. Brefeldin A (10 μg/mL, Sigma) was added for the last 4 h of culture. After incubation, intracellular staining was performed according to the manufacturer's instructions (BD Pharmingen). Antibodies used were as follows: anti-IFNγ-PE (Biolegend), anti-MHC II-APC/cy7 (Biolegend), anti-CD80-BV421 (Biolegend), anti-CD86-PE/cy7 (Biolegend), anti-CD3-FITC (Biolegend), anti-CD4-PerCP (Biolegend), anti-CD69-APC/cy7 (Biolegend), anti-IL-4-PE (Biolegend), anti-IL-5-BV421 (eBioscience), anti-IL-13-PE/cy7 (eBioscience), anti-CD45-APC (eBioscience), anti-ST2-PE (eBioscience), and FITC-conjugated anti-lineage antibodies (eBioscience). The lineage marker antibody cocktail was composed of antibodies against: DX5 (or NK1.1), CD3, CD4, CD5, CD8, CD11b, CD19, B220, Gr-1 and TCRδ (BD Bioscience).
Neutralizing Antibody: Anti-mouse MHC Class II (clone: M5/114; InVivoMAb grade; BioXcell, BE0108), anti-mouse CD4 (α-CD4; clone: GK1.5; InVivoMAb grade; BioXcell, BE0003).

Kang L., Wang S., Wang D., Wang J., Zheng R., Jiang X, & Liu B. (2022). Group 2 innate lymphoid cells mediate the activation of CD4⁺ T cells and aggravate Th1/Th2 imbalance via MHC II molecules during respiratory syncytial virus infection. International immunopharmacology, 113(Pt A).

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