Naive CD4+ T cells were purified by mouse CD4+CD62L+ T Cell Isolation Kit II (Miltenyi Biotec). Purified naive T cells were stimulated with plate-bound goat anti-hamster antibodies, soluble anti-CD3 (0.25 μg ml−1,145-2C11; Biolegend) and anti-CD28 (0.5 μg ml−1, 37.51; Biolegend) for Th0-non-polarized condition culture. In addition to Th0-non-polarized condition, following stimulation was used for each polarized condition: IL-12 (20 ng ml−1; R&D Systems) and anti-IL-4 (10 μg ml−1, C17.8; Biolegend) for Th1; IL-4 (100 ng ml−1; R&D Systems), anti-IL-12 (10 μg ml−1; Biolegend) and anti-IFNγ (10 μg ml−1, XMG1.2; Biolegend) for Th2; IL-6 (3 ng ml−1 or indicated concentration; R&D Systems), TGF-β1 (0.3 ng ml−1; R&D Systems), anti-IL-4 (10 μg ml−1, C17.8; Biolegend) and anti-IFNγ (10 μg ml−1; XMG1.2; Biolegend) for Th17; and IL-2 (20 ng ml−1; R&D Systems), TGF-β1 (3 ng ml−1; R&D Systems), anti-IL-4 (10 μg ml−1, C17.8; Biolegend) and anti-IFNγ (10 μg ml−1, XMG1.2; Biolegend) for Tregs. For signal transduction studies, STA21 (STAT3 inhibitor; Santa Cruz Biotechnology) and SMAD3 inhibitor (SIS3; Santa Cruz Biotechnology) were added to cultures on day 0.
Anti il 4
Manufactured by BioLegend
Sourced in United States
Anti-IL-4 is a laboratory reagent that binds to and neutralizes the cytokine interleukin-4 (IL-4). IL-4 is a signaling molecule that plays a role in the immune response.
Automatically generated - may contain errors
Lab products found in correlation
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106 protocols using anti il 4
1
Naïve CD4+ T Cell Polarization
2
Polarization of Naive CD4+ T Cells
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Naive CD4+ T cells were enriched from PBMCs by negative magnetic separation using MojoSort Human CD4 Naive T cell isolation Kit (BioLegend). The purity of isolated cells was checked by flow cytometry (> 95%). 2.5 × 105 naive cells were cultured in flat-bottom 96-well plates with CD3/CD28 Dynabeads in RPMI 1640 supplemented with 15% FCS, 10 mM HEPES (Sigma), 100 U/ml penicillin (Sigma), and 200 mM L-glutamine (Sigma) for 4 days under the following conditions: Th0 (anti-IL-4 (5 μg/ml), anti-IFNγ (5 μg/ml)); Th1 (anti-IL-4 (5 μg/ml), IL-12 (10 ng/ml)); Th2 (IL-4 (10 ng/ml), anti-IFNγ (5 μg/ml)); Th17 (IL-6 (20 ng/ml), TGFβ (1 ng/ml), anti-IFNγ (5 μg/ml), anti-IL-4 (5 μg/ml)) (all recombinant proteins and antibodies were from BioLegend). On day 4, cells were stimulated with PMA (50 ng/ml) and ionomycin (375 ng/ml) for 5 h. Golgi Plug was added into the culture (1:1000 dilution) 1 h after stimulation. The levels of IFNγ, IL-4, and IL-17A were measured by flow cytometry with intracellular staining protocol.
3
Multiparameter Flow Cytometry Analysis
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Human cells were stained with the following antibodies: fluorescein isothiocyanate (FITC)-anti-CD4, PE-cyanine 7 (Cy7)-conjugated anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend, San Diego, CA, USA). Mouse cells were stained with FITC-conjugated anti-CD4, PE-anti-chemokine (C-X-C) motif receptor 5, PE-Cy7-conjugated anti-inducible costimulatory molecule, anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend).
Intracellular staining was performed as follows. PBMCs or splenocytes were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate, 750 ng/ml ionomycin (both from Sigma-Aldrich), and 1 μl/ml GolgiStop (BD Biosciences) for 5 h at 37 °C. Surface staining was performed for 20 minutes with FITC anti-human/anti-mouse CD4 antibody on ice. Cells were washed and resuspended in fixation/permeabilization solution (BD Cytofix/Cytoperm kit; BD Biosciences) and stained with PE-Cy7-conjugated anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend) for flow cytometric analysis. PE-Cy7-conjugated IgG1 and FITC-conjugated IgG1 (BD Biosciences) were used as isotype controls. All data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
Intracellular staining was performed as follows. PBMCs or splenocytes were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate, 750 ng/ml ionomycin (both from Sigma-Aldrich), and 1 μl/ml GolgiStop (BD Biosciences) for 5 h at 37 °C. Surface staining was performed for 20 minutes with FITC anti-human/anti-mouse CD4 antibody on ice. Cells were washed and resuspended in fixation/permeabilization solution (BD Cytofix/Cytoperm kit; BD Biosciences) and stained with PE-Cy7-conjugated anti-IFN-γ, anti-IL-4, anti-IL-17A, and anti-TNF-α (all from BioLegend) for flow cytometric analysis. PE-Cy7-conjugated IgG1 and FITC-conjugated IgG1 (BD Biosciences) were used as isotype controls. All data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA).
4
In Vitro CD4+ T Cell Differentiation
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Naïve CD4+ T cells from the spleen and lymph nodes of 8–12 weeks C57BL/6 mice were purified by positive selection using CD4 conjugated magnetic beads (MiltenyiBiotec). CD4+ T cells (2 × 105/well) were cultured in vitro for 3 days (37 °C, 5% CO2) on flat- bottom 96- well plates coated with anti-CD3 (5 μg/ml) (145-2C11; Biolegend) and Anti-CD28 (1 μg/mL) (37.51; eBioscience) in the presence of tofacitinib, triamcinolone or NTG-A-009. OVA-specific naïve CD4+ T cells from spleen and lymph nodes of OT-II mice were purified by positive selection with CD4-conjugated magnetic beads (MiltenyiBiotec), for antigen specific Th cell differentiation. CD4+ T cells (2 × 105/well) and irradiated APCs (1 × 105/well) were co-cultured in the presence of CD4+ T cells and were induced to differentiate in to Th1 cells under the supplementation with IL-12 (10 ng/mL) (Biolegend) and anti-IL4 (5 μg/mL) (Biolegend); as a blocking antibody. For Th17 differentiation, TGF-β1 (1 ng/mL) (R&D system), IL-6 (10 ng/mL) (R&D system) plus anti-IL-4 (5 μg/mL) (Biolegend) and anti- IFN-γ (5 μg/mL) (Biolegend); for Treg differentiation, IL-2 (10 ng/ml) (Biolegend), TGF-β1 (10 ng/mL) (R&D system) plus anti-IL-4 (5 μg/mL) (Biolegend) and anti- IFN-γ (5 μg/mL) (Biolegend) and cultured in a cell incubator with 5% CO2 at 37 °C for 72 h.
5
Murine CD4+ T cell differentiation
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Single cell suspensions were prepared from the lymph node and spleen of C57BL/6 mice and CD4+ T cells were isolated using the CD4+ T Cell Isolation Kit (130-104-454, Miltenyi Biotec). Cells were cultured in RPMI medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol and 10,000 U/ml penicillin/streptomycin. Cells were activated with plate-bound anti-mouse CD3 (5 μg/ml; clone 145-2C11), and soluble anti-mouse CD28 (2 μg/ml; clone 37.51, eBioscience) in RPMI for four days in the following differentiation media:-
(a) for Th1 - 10 ng/ml IL-12 (200–12), 10 ng/ml IL-2, 5 µg/ml anti-IL-4 (504122, BioLegend);
(b) for Th17 - 50 ng/ml IL-6 (216–16), 10 ng/ml IL-1β (211-11B), 10 ng/ml IL-23 (200–23), 5 ng/ml TGF-β (100–21), 5 µg/ml anti-IL-4, 5 µg/ml anti-IFN-γ (505834, BioLegend);
(c) for Tregs - 10 ng/mL IL-2 (200-02, PeproTech) and 10 ng/mL TGF-β DMSO or the indicated concentration of PQS was added from day 1 of stimulation and cell populations were analysed by FACS after 4 days
(a) for Th1 - 10 ng/ml IL-12 (200–12), 10 ng/ml IL-2, 5 µg/ml anti-IL-4 (504122, BioLegend);
(b) for Th17 - 50 ng/ml IL-6 (216–16), 10 ng/ml IL-1β (211-11B), 10 ng/ml IL-23 (200–23), 5 ng/ml TGF-β (100–21), 5 µg/ml anti-IL-4, 5 µg/ml anti-IFN-γ (505834, BioLegend);
(c) for Tregs - 10 ng/mL IL-2 (200-02, PeproTech) and 10 ng/mL TGF-β DMSO or the indicated concentration of PQS was added from day 1 of stimulation and cell populations were analysed by FACS after 4 days
6
Naïve T-cell Polarization Assay
7
In Vitro T Cell Differentiation
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OT-2 T cells and OVA-loaded APCs were prepared as above. Naive OT-2 T cells (5×104) and purified OVA-loaded antigen-presenting cells (1×104) were co-cultured in 96-well round-bottom tissue culture plates under the control (medium alone) or Th polarizing conditions (23 (link)) as follows: Th1 with 10 ng/ml IL-12 (BioLegend) and 5 μg/ml anti-IL-4 (BioLegend); Th2 with 10 ng/ml IL-2 (BioLegend), 10 ng/ml IL-4 (BioLegend), and 5 μg/ml anti-IFN-γ (BioLegend); Th17: 2 ng/ml TGF-β (Peprotech, Rocky Hill, NJ), 10 ng/ml IL-1β (BioLegend), 20 ng/ml IL-6 (BioLegend), 5 μg/ml anti-IFN-γ, and 5 μg/ml anti-IL-4. Intracellular staining of Th cytokines IFN-γ (Th1), IL-4 (Th2), and IL-17A (Th17) were performed by flow cytometry following stimulation with PMA, ionomycin, and brefeldin A at 3 to 5 days after culture.
8
Murine T Cell Subset Differentiation
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Splenocytes were harvested from 7-week-old mice, and naïve CD4+ T cells (CD4+CD25−CD62Lhigh) were enriched with the Naïve CD4+ Isolation Kit (STEMCELL Technologies). T cells were cultured with RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l -glutamine, 1% Antibiotic-Antimycotic, 10 mM Hepes, and 50 μM β-mercaptoethanol (all from Gibco). The naïve T cells were activated with plate-bound anti-CD3 (5 mg/ml; BD Biosciences) and soluble anti-CD28 (2 mg/ml; BD Biosciences). The Treg cell differentiation condition was achieved by adding transforming growth factor–β1 (TGF-β1) (5 ng/ml; R&D Systems). The TH17 cell differentiation condition was achieved by adding TGF-β1 (10 ng/ml, R&D Systems), anti–IFN-γ (10 μg/ml; BioLegend), anti–IL-4 (5 μg/ml; BioLegend), IL-23A (20 ng/ml; R&D Systems), and IL-6 (10 ng/ml; R&D Systems). The TH1 cell differentiation condition was achieved by adding IL-12 (20 ng/ml; R&D Systems) and anti–IL-4 (10 μg/ml; BioLegend). All T cell subsets were cultured for 3 days and detected using flow cytometry.
9
In Vitro T Cell Differentiation
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CD4+ T cells were isolated from spleen and lymph nodes of mice by negative selection using the MACs separation system (Miltenyi Biotec). Cells were resuspended in complete RPMI and were plated with soluble anti-mouse CD28 antibody (2 μg/mL) on 96-well plates that were pre-coated with LEAF purified anti-mouse CD3 antibody (2 μg/mL) (both from BioLegend). For proliferation assays, cells were first labeled with 0.5 μM CFSE prior to culture. For Th differentiation assays, cells were stimulated without additional reagents (Th0 conditions), in the presence of 10 μg/mL anti-IL-4 (BioLegend) and 5 ng/mL recombinant mouse IL-12 (Peprotech) (Th1 conditions), or with 10 μg/mL anti-IL-4, 10 μg/mL anti-IFN-γ (BioLegend), 20 ng/mL recombinant mouse IL-6 (Peprotech), and 5 ng/mL recombinant human TGF-β (Prospec) (Th17 conditions). After 5 days, cells were re-stimulated with PMA and ionomycin and stained with antibodies specific for CD4, IFN-γ and IL-17 as described above.
10
Isolation and Differentiation of T Cell Subsets
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Naive CD4+ T cells (CD4+CD44lowCD62LhiCD25−) were isolated by MACS beads cell isolation kits (Miltenyi Biotec) or flow cytometry from the spleen using FACS ARIAII (BD). Naive CD4+ T cells were stimulated with plate-bound anti-CD3ε (5 µg/ml; 145-2C11; BioLegend) and anti-CD28 (2 µg/ml; 37.51; BioLegend), in the presence of IL-12 (20 ng/ml) and anti–IL-4 (10 µg/ml; 11B11; BioLegend) for the generation of Th1 cells; or IL-4 (50 ng/ml) and anti–IFN-γ (10 µg/ml; XMG1.2; BioLegend) for the generation of Th2 cells; or IL-6 (20 ng/ml), TGF-β1 (4 ng/ml), anti-IL4, and anti-IFN-γ for the generation of Th17 cells; or IL-2 (20 U/ml) and hTGF-β1 (4 ng/ml) for the generation of regulatory T cells. Mouse IL-4, IL-6, IL-12, IL-2, and human TGF-β1 were obtained from R&D Systems.
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