Anti il 17a clone tc11 18h10

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Anti-IL-17A (clone TC11-18H10.1) is a mouse monoclonal antibody that binds to human interleukin-17A (IL-17A). IL-17A is a pro-inflammatory cytokine involved in various immune responses.

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6 protocols using anti il 17a clone tc11 18h10

Cytokine Production Profiling Assay

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Cells were stimulated phorbol 12-myristate-13-acetate (PMA) (81 nM) and ionomycin (1.3 μM) (Biolegend) for one hour at 37°C and 5% CO₂ then monensin (2 μM) (Biolegend) was added without washing the cells for four additional hours. Cells were then washed with staining buffer, stained for surface receptors, then fixed for 20 minutes, and stained for intracellular cytokines in permeabilization buffer. The intracellular antibodies that were used were anti-TNF-α (clone MP6-XT22, Biolegend), anti-IL-6 (clone MP5-20F3, Biolegend), anti-IL-17A (clone TC11-18H10.1, Biolegend), anti-IFN-γ (clone XMG1.2, Biolegend), anti-IL-2 (clone JES6-5H4, Biolegend), anti-IL-4 (clone 11B11, Biolegend), and anti-IL-10 (clone JES5-16E3, Biolegend).
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII or Celesta (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR).

Muhammad F., Wang D., McDonald T., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2020). TIGIT+ A2Ar-Dependent Anti-Uveitic Treg Cells are a Novel Subset of Tregs Associated with Resolution of Autoimmune Uveitis. Journal of autoimmunity, 111, 102441.

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Multiparametric Immune Phenotyping of Cells

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Single cell suspensions were treated with Zombie violet as per the manufacturers insert instructions (Biolegend). Following treatment with Fc block (1 μg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145– 2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4–4), CD4 (clone RM 4–5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11–18H10.1) antibodies (BioLegend). Data was acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the supplementary data.

O’Hara J.M., Redhu N.S., Cheung E., Robertson N.G., Patik I., Sayed S.E., Thompson C.M., Herd M., Lucas K.B., Conaway E., Morton C.C., Farber D.L., Malley R, & Horwitz B.H. (2019). Generation of protective pneumococcal-specific nasal resident memory CD4+ T cells via parenteral immunization. Mucosal Immunology, 13(1), 172-182.

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Mouse Immune Cell Analysis

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Fluorochrome conjugated antibodies specific for mouse, anti-CD3 (clone: 145-2C11), anti-CD4 (clone: RM4-5), anti-Foxp3 (clone: MF-14), anti-IFNγ (clone: XMG1.2) and anti-IL-17A (clone: TC11-18H10.1) were all purchased from Biolegend, San Diego, CA, USA. Anti-CD8 (clone: 53–6.7) was purchased from eBioscience, Frankfurt, Germany.

Prajeeth C.K., Beineke A., Iskandar C.D., Gudi V., Herder V., Gerhauser I., Haist V., Teich R., Huehn J., Baumgärtner W, & Stangel M. (2014). Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice. Journal of Neuroinflammation, 11, 180.

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Multi-parameter flow cytometric analysis

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For surface staining anti-CD4 (clone GK1.5, eBioscience), anti-IL22R1 (clone 496514, R&D sytems) or relevant isotype control antibodies were used. For intracellular flowcytometry, splenocytes or paws cells were stimulated with PMA(5 ng/ml) and Ionomycin(500 ng/ml) (Sigma-Aldrich, USA) and Brefeldin A (Biolegend) for 6 hours, prior to staining with anti-IL-17A (clone TC11-18H10.1, Biolegend), anti-IFN-γ (clone XMG1.2, eBioscience), or anti-IL-22 (clone IL-22JOP, eBioscience) antibodies or relevant isotype controls. Data was acquired with BD LSR and analyzed using Flow-Jo software.

Justa S., Zhou X, & Sarkar S. (2014). Endogenous IL-22 Plays a Dual Role in Arthritis: Regulation of Established Arthritis via IFN-γ Responses. PLoS ONE, 9(3), e93279.

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Quantification of Antigen-Specific T Cell Responses

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To quantify antigen-specific cytokine-producing CD4⁺ and CD8⁺ cells, cells were restimulated as described, with the addition of 4.1 μg/mL Brefeldin A (BD Biosciences) for the last 6 hours of incubation. Restimulated splenocytes were collected, washed with 1 X PBS, and stained with Live/Dead fixable blue viability dye, anti-CD3e Pacific Blue clone 145-2C11 (Biolegend), anti-CD4 Alexa Fluor^® 700 clone RM4-5 (eBioscience) and anti CD8a PerCP-Cy5.5 clone 53-6.7 (BD Bioscience). Splenocytes were then fixed with Cytofix/Cytoperm (BD Biosciences) and permeabilized following manufacturer instructions. Permeabilized splenocytes were stained with anti-IFNγ APC clone XMG1.2 (eBioscience), anti-IL-17A clone TC11-18H10.1 (Biolegend), anti-IL-2 Brilliant Violet 510 clone JES6-5H4 (Biolegend), anti-IL-4 PE-Cyanine7 clone BVD6-24G2 (eBioscience), and anti-TNFα PE clone MP6-XT22 (eBioscience). Samples were acquired on a LSR Fortessa Cell Analyzer (BD Biosciences) and 25,000 total events in a live gate were analyzed using FlowJo 10.8.1 software. To evaluate antigen-specific responses the percent of unstimulated cells was subtracted from cells stimulated with Tc24-C4 protein for each sample.

Liu Z., Ulrich R., Kendricks A.L., Wheeler K., Leão A.C., Pollet J., Bottazzi M.E., Hotez P., Gusovsky F., Jones K.M, & McCall L.I. (2023). Localized cardiac metabolic trajectories and post-infectious metabolic sequelae in experimental Chagas disease. Research Square.

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Multiparameter Flow Cytometry Analysis

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Single-cell suspensions were treated with Zombie violet as per the manufacturer's insert instructions (Biolegend). Following treatment with Fc block (1 µg/ml) (Biolegend), cells were stained with combinations of the following antibodies: CD69 (clone H1.2F3), CD44 (clone IM7), CD8α (clone 53–6.7), CD3e (clone 145-2C11), CD3 (clone 17A2), CD11a (clone I21/7), CD45 (clone 30-F11), Thy1.2 (clone 53–2.1), CD4 (clone GK1.5), CD4 (clone RM 4-4), CD4 (clone RM 4-5), CD62L (clone MEL-14) (BioLegend). Intracellular cytokine staining was performed with anti-IFN-γ (clone XMG1.2) and anti-IL17A (clone TC11-18H10.1) antibodies (BioLegend). The data were acquired using a FACS Canto II flow cytometer (Becton Dickinson (BD)) or Attune NxT flow cytometer (ThermoFischer Scientific) and analyzed using FlowJo software (TreeStar, version 10). All gating stragies used are described in the Supplementary Data.

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