Anti il 4

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Anti-IL-4 is a laboratory reagent used in research applications. It is an antibody that targets the cytokine interleukin-4 (IL-4). The primary function of Anti-IL-4 is to enable the detection and measurement of IL-4 levels in biological samples.

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Close spelling variants of this product

Pe-Cy7-conjugated anti-IL-4 Anti-human IL-4 Anti-IL-4 (11B11) Anti-IL-4 antibody Anti-IL-4 mAb Purified anti-IL-4 antibody Neutralizing anti-IL-4 Anti-IL-4 neutralizing antibody Anti-IL-4-APC PE-conjugated anti-IL-4

121 protocols using anti il 4

Differentiation of Murine T Cell Subsets

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Naive CD4⁺CD25⁻CD62L^hiCD44^lo T cells from spleens and lymph nodes were isolated using a FACSAria sorter (BD Biosciences). Purified naive T cells were stimulated with 2 μg/mL plate-bound anti-CD3 (2C11, Bio X Cell) and 2 μg/mL anti-CD28 (37.51, Bio X Cell) in the presence of 5 μg/mL anti–IFN-γ (XMG1.2, Bio X Cell), 5 μg/mL anti–IL-4 (11B11, Bio X Cell), and 40 U/mL IL-2 (Peprotech) for the generation of Th0 cells; 20 ng/mL IL-12 (Peprotech), 5 μg/mL anti–IL-4, and 40 U/mL IL-2 for the generation of Th1 cells; 10 ng/mL IL-4 (Peprotech), 10 μg/mL anti–IFN-γ and 40 U/mL IL-2 for the generation of Th2 cells; 1 ng/mL TGF-β1 (Peprotech), 10 ng/mL IL-6 (Peprotech), 5 μg/mL anti–IFN-γ, and 5 μg/mL anti–IL-4 or 10 ng/mL IL-23, 10 ng/mL IL-1β (Peprotech), 10 ng/mL IL-6, 5 μg/mL anti–IFN-γ, and 5 μg/mL anti–IL-4 for the generation of Th17 cells; and 1 ng/mL TGF-β1, 5 μg/mL anti–IFN-γ, 5 μg/mL anti–IL-4, and 40 U/mL IL-2 for the generation of iTregs. Cells were cultured in complete medium (RPMI medium containing 10% FBS, supplemented with penicillin-streptomycin, HEPES, l-glutamine, sodium pyruvate, and 2-mercaptoethanol) for 3–5 days, followed by intracellular staining and RNA preparation.

Chen X., Hu J., Wang Y., Lee Y., Zhao X., Lu H., Zhu G., Wang H., Jiang Y., Liu F., Chen Y., Kim B.S., Zhou Q., Liu X., Wang X., Chang S.H, & Dong C. (2022). The FoxO4/DKK3 axis represses IFN-γ expression by Th1 cells and limits antimicrobial immunity. The Journal of Clinical Investigation, 132(18), e147566.

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Isolation and Polarization of Human Naive CD4+ T Cells

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As was described previously 28 (link), human peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood of healthy subjects and rosacea patients by Density gradient centrifugation using Ficoll-Paque (GE Healthcare). Naive CD4⁺ T cells were isolated from PBMCs by magnetic positive selection using the human naive CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec) and cultured in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco). Naive CD4⁺ T cells were activated for 5 days by following antibodies and cytokines in the presence of 5μg/ml anti-CD3 (Cat #217570, Calbiochem) and 2μg/ml anti-CD28 (Cat #217669, Calbiochem). The polarizing conditions: 10μg/ml anti-IFN-γ (Cat #16-7317-85, eBioscience) and 10μg/ml anti-IL-4 (Cat #16-7048-85, eBioscience) for Th0; 10μg/ml anti-IL-4 and 10ng/ml IL-12 (Cat #200-12, PeproTech) for Th1; 10μg/ml anti-IFN-γ and 2.5ng/ml IL-4 (Cat #214-04, PeproTech) for Th2; 10μg/ml anti-IFN-γ, 10μg/ml anti-IL-4, 5ng/ml TGF-β(Cat #100-21, PeproTech), 10ng/ml IL-6 (Cat #200-06, PeproTech), 10ng/ml IL-1β (Cat #200-01, PeproTech), and 20ng/ml IL-23 (Cat #200-23, PeproTech) for Th17; 5ng/ml TGF-βand 10ng/ml IL-2 (Cat #200-02, PeproTech) for iTreg. 1 × 10⁶ naive CD4⁺ T cells were seeded in 24-well plates and the medium was refreshed on the 3rd day.

Chen M., Peng Q., Tan Z., Xu S., Wang Y., Wu A., Xiao W., Wang Q., Xie H., Li J., Shi W, & Deng Z. (2023). Targeting Aquaporin-3 Attenuates Skin Inflammation in Rosacea. International Journal of Biological Sciences, 19(16), 5160-5173.

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CD4+ T Cell Differentiation Protocol

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CD4 T cells were purified from splenocytes using the Dynabeads^® FlowComp™ isolation kit (Invitrogen) according to manufacturer’s instructions. Purified CD4+ cells were plated with 3 μg/mL plate-bound anti-CD3ε (145–2C11, Biolegend) and 5 μg/mL soluble anti-CD28 (37.51, Bio-X-Cell) in complete RPMI. For differentiation the following were added: 1 ng/mL IL-12 (Peprotech) and 5 μg/mL anti-IL-4 (eBioscience) for Th1; 10 ng/mL IL-4 (Peprotech) and 5 μg/mL anti-IFNγ (Biolegend) for Th2; 2 ng/ml TGFβ (Biolegend), 50 U/ml IL-2 (Peprotech), 5 μg/ml anti-IFNγ and 5 μg/ml anti-IL-4 for iTreg; and 1 ng/mL TGFβ, 20 ng/ml IL-6 (Peprotech), 5 μg/ml anti-IFNγ and 5 μg/ml anti-IL-4 for Th17. Cells were re-stimulated with 20 ng/mL phorbol myristate acetate (PMA) and 400 ng/mL ionomycin, or plate-bound anti- CD3ε prior to intracellular cytokine staining or preparation of RNA. When cells were used for intracellular cytokine staining, they were also incubated with 1 μg/mL Brefeldin A (eBioscience) for 4 hours following an initial 2 hours of re-stimulation.

Lyon de Ana C., Arakcheeva K., Agnihotri P., Derosia N, & Winandy S. (2019). Lack of Ikaros deregulates inflammatory gene programs in T cells¶. Journal of immunology (Baltimore, Md. : 1950), 202(4), 1112-1123.

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Cytokine-Driven T Cell Differentiation

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Purified human or mouse naive CD4⁺ T cells were stimulated with plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) alone (Th0) or under Th1 (10 ng/mL IL-12 and 10 μg/mL anti-IL4, Peprotech, USA), Th2 (20 ng/mL IL-4 and 10 μg/mL anti-IFN-γ, Peprotech), Th17 (2.5 ng/mL TGF-β, 15 ng/mL IL-6, 10 μg/mL anti-IFN-γ and 10 μg/mL anti-IL4, Peprotech) and Treg (1.5 ng/mL TGF-β, 10 μg/mL anti-IFN-γ and 10 μg/mL anti-IL4, Peprotech) conditions. After 5 d of stimulation, the cells were subjected to Flow cytometry analysis, ELISA, or qPCR analyses.

Wang P., Zhao J., Tan Y., Sheng J., He S., Chen Y., Nie D., You X., Luo J., Zhang Y, & Hu S. (2023). RNF157 attenuates CD4+ T cell-mediated autoimmune response by promoting HDAC1 ubiquitination and degradation. Theranostics, 13(11), 3509-3523.

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Naive CD4+ T Cell Differentiation

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Naïve CD4+ T cells were isolated from the spleen of 6–8 w/o female mice by magnetic cell sorting (Biolegend, CA, USA). Magnetically sorted naïve CD4+ T cells were grown in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution at 37°C in an incubator with 5% CO₂. Plate-bound anti-CD3 (2 μg/mL, Biolegend) and soluble anti-CD28 (1 μg/mL, Biolegend) were used to activate naïve CD4+ T cells, followed by differentiation into Th1 (IL-12 [5 ng/mL; Peprotech, NJ, USA], IL-2 [(50 U/mL); Biolegend] and anti-IL-4 [10 μg/mL; Peprotech]); Th2 (IL-4 [20 ng/mL; Biolegend] and anti-IFN-γ [10 μg/mL; Biolegend]), Th9 (IL-4 [20 ng/mL], TGF-β [2 ng/mL; Biolegend] and anti-IFN-γ [10 μg/mL]), Th17 (TGF-β [2 ng/mL], IL-6 [100 ng/mL; Peprotech], IL-1β [10 ng/mL; Peprotech], IL-23 [10 ng/mL; Peprotech], anti-IFN-γ [10 μg/mL] and anti-IL-4 [10 μg/mL]) and Treg cell differentiating conditions (TGF-β [2 ng/mL], anti-IL-4 [10 μg/mL; Peprotech] for 3 days. The cultures were expanded for further 2 days by adding three times of fresh media for all the culture conditions with IL-4 and TGF-β for Th9, half the concentration of IL-6, IL-1β, IL-23 for Th17 and IL-2 for Treg conditions. Calcitriol used in the experiments was procured from Sigma Aldrich (MO, USA).

Vyas S.P., Hansda A.K., Kaplan M.H, & Goswami R. (2020). Calcitriol regulates the differentiation of IL-9-secreting Th9 cells by modulating the transcription factor PU.1. Journal of immunology (Baltimore, Md. : 1950), 204(5), 1201-1213.

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CD4+ T Cell Polarization Assay

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Purified naive CD4⁺ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 under Th1 (10 ng/ml IL-12 and 10 μg/ml anti-IL4, Peprotech, USA), Th2 (20 ng/ml IL-4 and 10 μg/ml anti-IFN-γ, Peprotech), Th17 (2.5 ng/ml TGF-β, 15 ng/ml IL-6, 10 μg/ml anti-IFN-γ, and 10 μg/ml anti-IL4, Peprotech), and Treg (1.5 ng/ml TGF-β, 10 μg/ml anti-IFN-γ, and 10 μg/ml anti-IL4, Peprotech) conditions. After 5 days of stimulation, the cells were subjected to ELISA or qPCR analyses.

Fu Y., Wang P., Zhao J., Tan Y., Sheng J., He S., Du X., Huang Y., Yang Y., Li J., Cai Y., Liu Y, & Hu S. (2021). USP12 promotes CD4+ T cell responses through deubiquitinating and stabilizing BCL10. Cell Death and Differentiation, 28(10), 2857-2870.

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In Vitro T Cell Polarization Assay

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CD11c⁺ DCs (10⁵ cells/ml) isolated from MLNs of mice in each groups were cultured for 72 h with CD4⁺CD62L^highCD44^low naïve T cells (10⁶ cells/ml) isolating from spleens of Tm-treated mice, in the presence of Tm (20 µg/ml) in RPMI 1640 culture medium (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% heat inactivated FBS (Tianhang, Hangzhou, China), 100 U/ml penicillin, 100 µg/ml streptomycin. T cells were polarized to Th1, Th2, Th17, and Treg subtypes in vitro by the addition of the following for 3 days: Th1, IL-12 (10 ng/ml; Peprotech, Rocky Hill, NJ, USA) and anti-IL-4 (10 µg/ml; eBioscience); Th2, IL-4 (10 ng/ml; Peprotech) and anti-IL-12 (10 µg/ml; eBioscience); Th17, TGF-β1 (0.2 ng/ml; Peprotech), IL-6 (40 ng/ml; Peprotech), anti-IFN-γ and anti-IL-4 (10 µg/ml; eBioscience); and Treg, TGF-β1 (0.2 ng/ml; Peprotech) and IL-2 (10 ng/ml; Peprotech). For APC-free T cell differentiation, plates were coated with anti-CD3 and anti-CD28 (10 µg/ml; eBioscience) at 4°C overnight, and then T cell with/without Binf (10⁵ CFU/ml) were differentiated to Th1, Th2, Th17, and Treg subtypes for 3 days with the same stimulators as in the DC-mediated polarization. After cultivation, precipitated cells were lysed and further used for RT-qPCR and ELISA analysis.

Fu L., Song J., Wang C., Fu S, & Wang Y. (2017). Bifidobacterium infantis Potentially Alleviates Shrimp Tropomyosin-Induced Allergy by Tolerogenic Dendritic Cell-Dependent Induction of Regulatory T Cells and Alterations in Gut Microbiota. Frontiers in Immunology, 8, 1536.

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Isolation and Polarization of Th1 and Th17 Cells

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Naïve CD4 T cells were isolated from spleens using the Naive CD4⁺ T Cell Isolation Kit (130–104–453, Miltenyi Biotec). Next, 96-well plates were activated overnight with plate-bound mouse anti-CD3 at 5 μg/ml (BD Pharmingen Clone 145-C11 cat. no. 553058) and mouse anti-CD28 at 2 μg/ml (BD Pharmingen Clone 37.51 cat. no. 553295) at 4 °C. Purified CD4⁺ T cells (2 × 10⁵) were cultured 200 μl cDMEM in the activated plate. To induce Th1 polarization in vitro, the media was supplemented with anti-IL-4 (10 μg/ml) (ebioscience clone 11B11 cat. no. 16–7041–85) and IL-12 (20 ng/ml) (R&D Systems cat. no. 419-ML-010). Th1 cells were used at 4–6 days polarization.
For Th17 polarization, T-cell media was supplemented with Th17-polarizing cytokines: IL-23 (10 ng/ml, Protein R&D Systems, 1886-ML), IL-6 (100 ng/ml, Protein R&D Systems, 406-ML/CF), rTGF-β1 (2 ng/ml, Protein R&D Systems, 7666-MB-005), anti-IL-4 (10 μg/ml, eBioscience, 16–7041–85), and anti-IFN-γ (10 μg/ml, eBioscience 16–7312–85). Th17 cells were ready for analysis on day 5 of polarization.

Liu Y., Bockermann R., Hadi M., Safari I., Carrion B., Kveiborg M, & Issazadeh-Navikas S. (2020). ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation. Cellular and Molecular Immunology, 18(8), 1904-1919.

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In Vitro T Helper Cell Differentiation

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CD4⁺ T cells were isolated from single-cell suspensions of spleens and lymph nodes and purified by negative separation with magnetic beads (Stemcell). For in vitro differentiation into Th1, Th17 and iTreg cells, CD4⁺ T cells were stimulated with hamster anti-CD3 and hamster anti-CD28 together with 10 ng/ml IL-12 (Peprotech) and 2 μg/ml anti-IL-4 (eBioscience) for Th1, 20 ng/ml IL-6 (Peprotech), 0.5 ng/ml human TGF-β1 (Peprotech), 2 μg/ml anti-IL-4 and 2 μg/ml anti-IFN-γ (eBioscience) for Th17 and 2.5 ng/ml TGF-β for iTreg cells in IMDM medium (Cellgro) containing 2 mM L-Glutamine, 50 μM β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS in anti-hamster IgG coated plates for 3 days.

Kaufmann U., Shaw P.J., Kozhaya L., Subramanian R., Gaida K., Unutmaz D., McBride H.J, & Feske S. (2015). Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function. Journal of immunology (Baltimore, Md. : 1950), 196(2), 573-585.

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In Vitro T Helper Cell Skewing

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T_H17 and T_H1 skewing in vitro was performed by using total lymphocytes or selecting CD4⁺ T cells from spleens and lymph nodes of 4-week old mice (Miltenyi Biotec). The cells were skewed towards the T_H17 lineage for 4 days on 1µg/ml anti-CD3 and 2µg/ml anti-CD28 coated plates along with 0.3ng/ml TGF-β1 (R&D Systems), 20ng/ml IL-6 (R&D Systems), 10ng/ml IL-23 (eBioscience), 10µg/ml anti-IL4 (eBioscience), and 10µg/ml anti-IFNγ (eBioscience) in IMDM supplemented with 10% FBS, 50µM β-Mercaptoethanol, 2-mM L-glutamine, non-essential amino acids, 1 mM sodium pyruvate, and 10 mM Hepes. After 4 days, cells were collected for analysis. Cells analyzed by intracellular cytokine staining were stimulated with 50 ng/ml PMA and 1 µM Ionomycin along with GolgiStop (BD Pharmingen) for 5 hours prior to staining. Intracellular staining for IL-17A (BD Pharmingen) and IFNγ (eBioscience) was performed by fixing the cells in 4% paraformaldehyde followed by permeabilization with 0.1% Saponin. T_H1 skewing was performed by culturing total lymphocytes or CD4⁺ cells on 1µg/ml anti-CD3 and 2 µg/ml anti-CD28 coated plates along with 100U/ml IL-2 (Peprotech), 10ng/ml IL-12 (Ebiosciences), and 10 µg/ml anti-IL4 (eBioscience) and analysis was performed on day 7 as described above for T_H17 cells.

Buckley M.W., Trampont P.C., Arandjelovic S., Fond A.M., Juncadella I.J, & Ravichandran K.S. (2015). ShcA regulates late stages of T cell development and peripheral CD4+ T cell numbers. Journal of immunology (Baltimore, Md. : 1950), 194(4), 1665-1676.

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