Anti il 4

Manufactured by Cytek Biosciences

Anti-IL-4 is a laboratory reagent used in flow cytometry and other immunoassays. It is a monoclonal antibody that binds to the cytokine interleukin-4 (IL-4), a key regulator of immune responses. The core function of Anti-IL-4 is to detect and quantify IL-4 levels in biological samples.

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3 protocols using anti il 4

Isolation and Polarization of Murine T Cell Subsets

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Naïve CD4⁺ T cells were isolated and purified from the spleens of C57BL/6 mice using a naïve CD4⁺ T cell isolation kit (Stemcell Technologies, Cat # 19765), and then cultured in 96-well round bottom plates with 0.5 μg/mL plate-bound anti-CD3 (Biolegend, Cat # 100340). T cells were cultured in RPMI medium (ThermoFisher Scientific, Cat #11879020) supplemented with indicated concentrations of glucose and 10% fetal bovine serum. Inducible Treg (iTreg) polarizing cultures also contained 1 μg/mL anti-CD28 (Biolegend, Cat # 102116), and either 15 ng/mL latent TGF-β (R&D Systems, Cat # 299-LT-005) or 0.5 ng/mL active TGF-β (PeproTech, Cat # 100–21). Th1 polarizing cultures contained 1 μg/mL anti-CD28, 5 μg/mL anti-IL-4 (Tonbo Biosciences, Cat # 70–7041), 3 ng/mL IL-12 (PeproTech, Cat # 210–12), and 5 ng/mL IL-2 (PeproTech, Cat # 212–12). Mixed T cell cultures contained 1 μg/mL anti-CD28, 0.5 ng/mL active or 15 ng/mL latent TGF-β, and 3 ng/mL IL-12. As indicated, some cultures included 15 mM lactate (Sigma-Aldrich, Cat # L4263) or 20 μM MCT1/2 inhibitor (Tocris Bioscience, Cat # 4960). Th17 differentiation was carried out with 1 μg/mL anti-CD28, 5 ng/mL active TGF-β, and 50 ng/mL IL-6.

Pitmon E., Meehan E.V., Ahmadi E., Adler A.J, & Wang K. (2023). High glucose promotes regulatory T cell differentiation. PLOS ONE, 18(2), e0280916.

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T-Cell Differentiation Experiments

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T-cell differentiation experiments were performed as described previously.^E8 Briefly, FoxP3- CD4+ T cells were purified and activated using anti-CD3/CD28 beads (Dynabeads, Invitrogen). For T_H1 differentiation, 1 × 10⁵ T cells were stimulated in the presence of IL-2 (20 U/mL; eBioscience), IL-12 (20 ng/mL; Tonbo), and anti–IL-4 (10 μg/mL; Tonbo). For Treg-cell differentiation, cells were stimulated in the presence of IL-2 (100 U/mL; eBioscience), TGF-β (5 ng/mL; Tonbo), and anti–IFN-γ (10 μg/mL; Tonbo). After 4 days, T cells were stimulated with ionomycin (500 ng/mL) and phorbol 12-myristate 13-acetate (50 ng/mL) in the presence of monensin (eBioscience) for 4 hours before intracellular staining. FoxP3 staining was performed using FoxP3/transcription factor staining kit (Tonbo).

Ku M., Ke E., Sabouri-Ghomi M., Abadejos J.R., Freeman B., Nham A., Phillips N., Yang K.Y., Lui K.O, & Kirak O. (2018). Deconstructive somatic cell nuclear transfer reveals novel regulatory T-cell subsets. The Journal of allergy and clinical immunology, 142(3), 997-1000.e4.

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Isolation and Culture of Naive T Cell Subsets

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T cells were enriched from spleens and LNs using the MagniSort CD4 negative selection kit (Thermo Fisher Scientific). Naive CD4⁺ T cells were isolated by flow cytometry on the basis of markers CD4⁺CD62L⁺CD44⁻CD25⁻ or using the EasySep mouse naive T cell isolation kit. 10⁵ naive T cells were cultured for 4 d (Th17 and induced T reg [iT reg] cells) in a 96-well flat-bottom plate coated with 2 µg/ml anti-CD3 (clone 2C11; Tonbo Biosciences) and 2 µg/ml anti-CD28 (clone 37.51; Tonbo Biosciences) with the relevant cytokines and blocking antibodies: classical Th17 (20 ng/ml IL-6, 2 ng/ml TGFβ, 10 µg/ml anti-IL4 [clone 11B11; Tonbo Biosciences], and 10 µg/ml anti-IFNγ [clone XMG1.2; Tonbo Biosciences]), pathogenic Th17 (20 ng/ml IL-6, 20 ng/ml IL-1β, 20 ng/ml IL-23, 10 µg/ml anti-IL4, and 10 µg/ml anti-IFNγ), iT reg cells (20 ng/ml TGFβ and 100 U/ml IL-2), or Th0 cells (100 U/ml IL-2). Th17 cell cultures were performed in Iscove’s medium, and iT reg cell cultures were performed in RPMI medium. All media were supplemented with 10% FBS, penicillin/streptomycin, glucose, pyruvate, β-mercaptoethanol, and Hepes. Cytokines were purchased from R&D Systems (murine IL-6 and human IL-2), Miltenyi Biotec (murine IL-1β and murine IL-23), or ProteinTech (HumanKine; human TGFβ).

Warshauer J.T., Belk J.A., Chan A.Y., Wang J., Gupta A.R., Shi Q., Skartsis N., Peng Y., Phipps J.D., Acenas D., Smith J.A., Tamaki S.J., Tang Q., Gardner J.M., Satpathy A.T, & Anderson M.S. (2021). A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance. The Journal of Experimental Medicine, 218(8), e20210759.

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