Anti il 5

Manufactured by BioLegend

Sourced in United States

Anti-IL-5 is a lab equipment product that specifically binds to and detects interleukin-5 (IL-5), a cytokine involved in the regulation of eosinophil function. This product can be used in various immunological and biochemical applications to measure IL-5 levels.

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4 protocols using anti il 5

Multiparametric Flow Cytometry Analysis

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All cells were labeled with live/dead dye near infra red (Life Technologies) for dead cell exclusion and treated with Fc Block (Miltenyi, San Diego, CA, USA) prior to staining with fluorescently labeled antibodies. Anti-human antibodies used for DC staining were anti-CD83 PE (Biolegend, San Diego, CA, USA), anti-CD86-PE-Cy7 (Biolegend), anti-HLA-DR V450 (BD), and anti-CD11c APC (BD). Antibodies used in the T cell characterization were anti-LAG-3 FITC (Novus, Littleton, CO, USA), anti-PD-1 PerCP-Cy5.5, anti-CD3 Alexa700, anti-CD4 Brilliant Violet 650, anti-CD8 Brilliant Violet 570, anti-IFN-γ PE, anti-IL-5, anti-CD62L PE, and anti-CD45RA Alexa700 (all, Biolegend), anti-CD25 PE, and anti-IFN-γ PE-Cy7, anti-IL-4 PE-Cy7 (all BD), and anti-TIM-3 APC (R&D Systems). Surface staining and intracellular staining was performed using Cytofix/Cytoperm™ Plus kit (BD) according to the manufacturer’s instructions. Data acquisition was performed using the LSRFortessa flow cytometer (BD). Data analysis was performed with FlowJo version 9 (Tree Star, Inc., Ashland, OR, USA).

Sabins N.C., Harman B.C., Barone L.R., Shen S, & Santulli-Marotto S. (2016). Differential Expression of Immune Checkpoint Modulators on In Vitro Primed CD4+ and CD8+ T Cells. Frontiers in Immunology, 7, 221.

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T Cell Cytokine Profiling in IRF5 Knockout Mice

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Freshly isolated spleen-derived DO11.10 CD4⁺ T cells from IRF5^+/+, IRF5^+/−or IRF5^−/−mice at 0.2×10⁶ cells/well on 24 well plates were stimulated with 5 μg/ml anti-CD3 (145–2C11, Bio X Cell, West Lebanon, NH) and 1 μg/ml anti-CD28 (PV-1, Bio X Cell) antibody-coated wells for 72 hours. The following were assessed by ELISA: from BD biosciences: anti-IL2, biotin anti-IL2, anti-IFNγ, biotin anti-IFNγ, anti-TGFβ, biotin anti-TGFβ; from ThermoFisher Scientific: anti-IL17, biotin anti-IL17, biotin anti-IL5, anti-IL13, biotin anti-IL13; from BioLegend: anti-IL4, biotin anti-IL4, anti-IL5, anti-IL10, biotin anti-IL10. MTT assay as per manufacture instructions (MilliporeSigma).

Yan J., Pandey S.P., Barnes B.J., Turner J.R, & Abraham C. (2020). T Cell-Intrinsic IRF5 Regulates T Cell Signaling, Migration, and Differentiation and Promotes Intestinal Inflammation. Cell reports, 31(13), 107820.

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Flow Cytometry Analysis of Immune Cells

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Abs for FACS analysis, Fcγ receptor-blocking mAb (CD16/32; 2.4G2), FITC-conjugated anti-γδTCR (GL-3), anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD25 (3C7), anti-Foxp3 (MF23), anti-IL-10 (JES5-16E3), anti-IL-17A (TC11-18H10.1), anti-IL-4 (11B11) and anti-IL-2 (JES6-5H4); APC-conjugated anti-CD44 (IM7) and anti-CD3ε (145-2C11); PE-conjugated anti-CD8α (53-6.7), anti-CD25 (3C7), anti-NK1.1 (PK136), anti-IFN-γ (XMG1.2), anti-IL-10 (JES5-16E3) and anti-IL-5 (TRFK5); PerCP-conjugated anti-CD4 (RM4-5) and anti-CD8α (53-6.7) mAbs were purchased from Biolegend (San Diego, CA). Purified anti-CD3 and anti-CD28 (37.51) mAbs were obtained from BD Biosciences, (San Diego, CA).

Wang Y., Jiang X., Zhu J., Dan Y.u.e., Zhang X., Wang X., You Y., Wang B., Xu Y., Lu C., Sun X, & Yoshikai Y. (2016). IL-21/IL-21R signaling suppresses intestinal inflammation induced by DSS through regulation of Th responses in lamina propria in mice. Scientific Reports, 6, 31881.

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Cytokine and Transcription Factor Analysis in Immune Cells

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For measurement of intracellular cytokine expression, cells were isolated ex vivo and stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin, in the presence of GolgiStop (BD Biosciences) for 3 h. Dead cells were excluded by Fixable Viability Dye eFluor 450 (eBioscience). Cells were stained with antibodies to surface antigens, and then fixed and permeabilized with the Cytofix/Cytoperm kit (BD Biosciences), followed by staining with anti-IL-5 (Biolegend) and anti-IL-13 (eBioscience).
For analysis of transcription factor expression, cells were stained with antibodies to surface antigens, then fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions, and stained with anti-GATA3 (eBioscience), anti-RORγt, anti-T-bet (BD Biosciences), or anti-Ki-67 (eBioscience).
For measurement of intracellular HIF1α, Lin⁻ bone marrow cells were cultured in complete media with 10 ng/ml IL-7 (Peprotech) and 10 ng/ml IL-33 (R&D Systems) for 4 days. Cells were collected and stained with antibodies to surface antigens, fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions, and then stained with anti-HIF1α (R&D Systems).

Li Q., Li D., Zhang X., Wan Q., Zhang W., Zheng M., Zou L., Elly C., Lee J.H, & Liu Y.C. (2018). The E3 ligase VHL promotes group 2 innate lymphoid cell maturation and function via inhibition of glycolysis and induction of interleukin 33 receptor. Immunity, 48(2), 258-270.e5.

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