Recombinant il 4

Manufactured by R&D Systems

Sourced in United States

Recombinant IL-4 is a cytokine produced in a laboratory setting. It is an interleukin-4 protein, which is involved in various immune system functions.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Il 13, by R&D Systems (2 mentions) Recombinant ifn γ, by R&D Systems (2 mentions) Recombinant il 13, by R&D Systems (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Magnetic microbead, by Miltenyi Biotec (1 mentions) P stat6, by Santa Cruz Biotechnology (1 mentions) P gsk3β, by Cell Signaling Technology (1 mentions) E cadherin, by BD (1 mentions) Stat6, by Santa Cruz Biotechnology (1 mentions) Ang 2, by Santa Cruz Biotechnology (1 mentions) Twist, by Abcam (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Ly294002, by Merck Group (1 mentions) Sp600125, by Merck Group (1 mentions) Horse serum, by Thermo Fisher Scientific (1 mentions)

19 protocols using recombinant il 4

Macrophage Polarization by ucMSCs

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Macrophages from the BAL fluid samples were cultured in 48-well plates (1 × 10⁴ cells/well) for 6h and stimulated with 20 ng/mL of recombinant IL-4 (R&D Systems, Minneapolis, MN, USA) for 42h. Four groups were set up for ex vivo experiments: PBS (control group), ucMSC (ucMSC-treated group), IL-4 (IL-4-stimulated group), and IL-4 + ucMSC (IL-4-stimulated group treated with ucMSC). ucMSCs (1 × 10⁴ cells/well) were treated for 18h after stimulation with recombinant IL-4.
Similar to ex vivo experiments, in vitro study was conducted using the Transwell Permeable Support (Costar, Kennebunk, ME, USA). The MH-S (ATCC, Manassas, VA, USA) was cultured in 48-well plates (1 × 10⁴) and stimulated with recombinant IL-4 (R&D systems). Four groups were set up for the experiments: PBS (control group), IL-4 (IL-4-stimulated group), IL-4 + DucMSC (IL-4-stimulated group directly pretreated with ucMSC), and IL-4 + TucMSC (IL-4-stimulated group indirectly treated with ucMSC via transwell system).

Mo Y., Kang H., Bang J.Y., Shin J.W., Kim H.Y., Cho S.H, & Kang H.R. (2022). Intratracheal administration of mesenchymal stem cells modulates lung macrophage polarization and exerts anti-asthmatic effects. Scientific Reports, 12, 11728.

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Generation of Immature Dendritic Cells

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Healthy blood donors that volunteered were HLA-typed by the tissue typing laboratory of the Charité, Campus Virchow Klinikum (Berlin, Germany). DCs were generated as described previously [10 ]. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated via Fi-coll density gradient centrifugation. CD14⁺ monocytes were enriched by using magnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany), and 3 × 10⁶ cells/well were cultured in six-well culture plates (TPP) in complete RPMI medium, containing 2 mM L-glutamine, 10 mM HEPES (Gibco), penicillin (100 U/ml), streptomycin (100 μg/ml), 10% FCS, 1000 U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) (sargramostim, Leukine^®, Berlex, Richmond, CA), and 100 U/ml recombinant IL-4 (R&D Systems, Wiesbaden-Nordenstadt, Germany). Fresh medium supplemented with cytokines was added every second day to the wells, and cells were harvested between day 6 and day 8. Immature DCs displayed a down-regulated CD14 expression and were further defined by the expression of high levels of CD11b and low levels of CD80 [10 ].

Fehlings M., Drobbe L., Beigier-Bompadre M., Viveros P.R., Moos V., Schneider T., Meyer T.F., Aebischer T, & Ignatius R. (2016). Usage of Murine T-cell Hybridoma Cells as Responder Cells Reveals Interference of Helicobacter Pylori with Human Dendritic Cell-mediated Antigen Presentation. European Journal of Microbiology & Immunology, 6(4), 306-311.

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Immunoblotting of EMT Markers

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Polyclonal antibodies against p-Akt (Ser473), Akt, and p-GSK3β, and monoclonal antibodies to β-catenin and Vimentin, were purchased from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibodies to JNK, Slug, Sox2, p-Stat6, Stat6, Ang-2, and VEGF, and monoclonal antibodies to p-JNK, β-actin, and Oct4 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal antibody to Snail was purchased from Novus Biologicals (Littleton, CO, USA). The monoclonal antibody E-cadherin was obtained from BD Transduction Laboratories (San Jose, CA, USA). The polyclonal antibody Twist was purchased from Abcam Inc. (Cambridge, MA, USA). Monoclonal antibodies to IL-4 and IL-4Rα were obtained from R&D systems (Minneapolis, MN, USA). Anti-mouse and anti-rabbit Alexa Fluor 488 and 555-conjugated secondary antibodies were purchased from Thermo Fisher scientific (Waltham, MA, USA). Inhibitors of JAK, PI3K (LY294002), and JNK (SP600125) were purchased from Merck Millipore (Darmstadt, Germany). Recombinant IL-4 was obtained from R&D systems (Minneapolis, MN, USA).

Kim E.S., Choi Y.E., Hwang S.J., Han Y.H., Park M.J, & Bae I.H. (2016). IL-4, a direct target of miR-340/429, is involved in radiation-induced aggressive tumor behavior in human carcinoma cells. Oncotarget, 7(52), 86836-86856.

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C2C12 Myoblast Differentiation and IL-4 Treatment

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C2C12 myoblast cells (ATCC, Manassas, VA, USA) were maintained in growth medium (GM) consisting of Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies Inc., Carlsbad, CA, USA) supplemented with 10% bovine growth serum (Cytiva, Tokyo, Japan) and 1% penicillin–streptomycin (Life Technologies Inc.). To induce the differentiation of myoblast cells, GM was replaced with differentiation medium (DM), DMEM supplemented with 2% horse serum (Life Technologies Inc.) and 1% penicillin–streptomycin.
Recombinant IL-4 (R&D Systems, Minneapolis, MN, USA) was used as previously described [25 (link),26 (link)]. IL-4 was dissolved in distilled water containing 0.1% bovine serum albumin (BSA; Wako, Tokyo, Japan) and added to the culture medium at a final concentration of 1 to 100 ng/mL.

Kurosaka M., Hung Y.L., Machida S, & Kohda K. (2023). IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger. Cells, 12(9), 1284.

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Cytokine Quantification in Biological Samples

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Supernatants collected from re-stimulated lymph node cells or BAL samples were added to Nunc Maxisorp 96-well plates (Thermo Fisher Scientific), which had been coated with purified anti-mouse IL-4 antibody (Thermo Fisher Scientific), IL-5 (BD Biosciences), IL-13 (Thermo Fisher Scientific) and IFN-γ (Thermo Fisher Scientific) all at 2 μg/ml O/N at 4 °C. Plates were subsequently blocked with 1% BSA. Cytokines were detected with biotinylated anti-mouse IL-4 (Thermo Fisher Scientific), IL-5 (Thermo Fisher Scientific), IL-13 (Thermo Fisher Scientific) and IFN-γ (Thermo Fisher Scientific) all at 0.5 μg/ml. Recombinant IL-4, IL-5, IL-13 and IFN-γ (R&D systems) were used as a standard (starting concentration 10 ng/ml). Antibody binding was detected with streptavidin-HRP (BD Biosciences) and developed with TMB substrate set (BD Biosciences).

Agaronyan K., Sharma L., Vaidyanathan B., Glenn K., Yu S., Annicelli C., Wiggen T.D., Penningroth M.R., Hunter R.C., Dela C.S, & Medzhitov R. (2022). Tissue remodeling by an opportunistic pathogen triggers allergic inflammation. Immunity, 55(5), 895-911.e10.

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Polarization of Macrophage Subtypes

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Type I subset and M2 macrophages were polarized as described previously (29 (link)). Primary BMDM macrophages were polarized toward the M1 phenotype with recombinant IFN-γ (100 U/ml, R&D Systems 485-ML) and/or LPS (100 ng/ml, L6529; strain 055:B5; Sigma-Aldrich, St. Louis, MO, USA) or toward the M2 phenotype with recombinant IL-4 (R&D Systems 404-ML) or recombinant IL-13 (R&D Systems 413-ML) (10 ng/ml) for the specified times under normal culture conditions. Unpolarized cells (M0) served as controls. BMDMs were washed with PBS and harvested for total RNA isolation.

Das A., Yang C.S., Arifuzzaman S., Kim S., Kim S.Y., Jung K.H., Lee Y.S, & Chai Y.G. (2018). High-Resolution Mapping and Dynamics of the Transcriptome, Transcription Factors, and Transcription Co-Factor Networks in Classically and Alternatively Activated Macrophages. Frontiers in Immunology, 9, 22.

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Isolation and differentiation of PBMC

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Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood samples by gradient centrifugation according to the protocol provided by GE‐Healthcare (Ficoll‐Paque PLUS). B cells (CD19⁺) and monocytes (CD14⁺, CD16^‐) were isolated from PBMC by negative selection using the EasyEights™ EasySep™ Magnet (18103), the EasySep™ Human B Cell Isolation Kit (17954), and the EasySep™ Human Monocyte Isolation Kit (19359) all provided by StemCell™ Technologies.
B cells were maintained for up to 2 days in Roswell Park Memorial Institute medium (RPMI) 1640 medium (R8758, Roswell Park Memorial Institute medium; Sigma‐Aldrich), supplemented with 10% fetal bovine serum (FBS, 10270–106), 1% antibiotic‐antimycotic solution (AA, 15240062), and 1% l‐glutamine solution (l‐Gln, 11500626) all provided by Gibco, Thermo Fisher Scientific.
Monocytes were seeded and differentiated to dendritic cells (DC) in complete RPMI 1640 medium containing 10% FBS, 1% AA, 1% l‐Gln and supplemented with 800 IU/ml recombinant GM‐CSF (Granulocyte macrophage colony‐stimulating factor) and 500 IU/ml recombinant IL‐4 (R&D Systems) for 6 days at 37°C and 5% CO₂.

Aliu B., Demeestere D., Seydoux E., Boucraut J., Delmont E., Brodovitch A., Oberholzer T., Attarian S., Théaudin M., Tsouni P., Kuntzer T., Derfuss T., Steck A.J., Ernst B., Herrendorff R, & Hänggi P. (2020). Selective inhibition of anti‐MAG IgM autoantibody binding to myelin by an antigen‐specific glycopolymer. Journal of Neurochemistry, 154(5), 486-501.

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Notch Signaling Pathway Evaluation

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Penicillin, streptomycin, tetracycline, Dulbecco’s Modified Eagle’s medium, and fetal bovine serum were obtained from Invitrogen (Life Technologies, Carlsbad, CA, USA). The protein assay kit was from Bio-Rad (Hercules, CA, USA). Fibronectin and S100A4 were from Sigma-Aldrich (St. Louis, MO, USA); CD31 antibodies were from BD Biosciences (BD Biosciences, San Jose, CA, USA); antibodies against Jagged 1, and Hes 1, Hey 1, and Hes 5 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Notch 1, N1ICD, RBP-Jκ, and iNOS were from Cell Signaling Technology (Danvers, MA, USA). The fluorescent-700/800 secondary antibodies were obtained from Invitrogen (Carlsbad, CA, USA), an antibody against CD45 was from Millipore (Billerica, MA, USA), and antibodies against F4/80, proliferating cell nuclear antigen, GFP, and rabbit anti-α-SMA were from Abcam (Cambridge, MA, USA). The GFP antibody was purchased from Rockland Immunochemicals (Limerick, PA, USA), and lipopolysaccharide and recombinant IL-4 were purchased from R&D Systems (Minneapolis, MN, USA). BrdU Labeling and Detection Kits were obtained from Roche (Indianapolis, IN, USA).

Wu Y., Liang M., Huang F., Cheng O.H., Xiao X., Lee T.H., Truong L, & Cheng J. (2023). Notch Blockade Specifically in Bone Marrow-Derived FSP-1-Positive Cells Ameliorates Renal Fibrosis. Cells, 12(2), 214.

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Naïve T Cell Differentiation Protocol

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Naïve CD4+CD25− T cells were isolated ex vivo from the spleens of the mice described above by magnetic microbead negative selection with >98% purity (Miltenyi #130-104-454). The isolated CD4+CD25− T cells were then cultured on irradiated splenic feeder cells (300 Gy) at a ratio of 1:5 (200,000 T cells:1 million irradiated splenocytes in a 24-well plate) in RPMI supplemented with 10% FBS, sodium pyruvate, penicillin/streptomycin, HEPES and beta-mercaptoethanol and 2.5μg/mL of soluble anti-CD3 antibody (eBioscience). The naïve T cells were then differentiated in vitro by adding the following cytokines to each generate each subset: Th1, 10ng/mL IL-12 (R&D Systems), 10μg/mL anti-IL-4 (eBioscience, clone 11B11), 1μg/mL anti-IFNγ (eBioscience); Th2, 1000U/mL recombinant IL-4 (R&D Systems), 10μg/mL anti-IL-12 (eBioscience), 10μg/mL anti-IFNγ; Th17, 20ng/mL IL-6 (R&D Systems), 2.5ng/mL TGFβ (R&D Systems), 10μg/mL anti-IFNγ; Treg, 3ng/mL TGFβ. On day 3 post stimulation, cells were split 1:2 and expanded with IL-2 for an additional 2 days (30 (link), 31 (link)). Cells were then harvested, washed 1x with PBS, pelleted and snap frozen.

Wu H., Herr D., MacIver N.J., Rathmell J.C, & Gerriets V.A. (2020). CD4 T Cells Differentially Express Cellular Machinery for Serotonin Signaling, Synthesis, and Metabolism. International immunopharmacology, 88, 106922.

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Isolation and Culture of Primary Murine Microglia

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Primary microglia cultures were obtained from P2 C57Bl/6 mice. Briefly, we removed meninges from each brain in cold KRB medium containing albumin 0.3% (Sigma) and MgCl2 0.04% (Sigma). Cortices were minced and incubated in 10 ml of KRB including Trypsin 0.25 mg/ml (Sigma) for 15 min at 37 °C, then we added 10 ml of complete KRB supplemented with DNase I 0.05 mg/ml (Sigma) and SB-Trypsin inhibitor 0.08 mg/ml (Sigma) to stop the reaction. Cells (3 × 10⁶/T75 flask) were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), L-glutamine (1 mM) (Gibco), penicillin (100 U/ml), (Gibco) and streptomycin (100 mg/ml), (Gibco) for 12 days. Microglia were shaken off and 3 × 10⁵ cells plated on glass coverslips. Cells were further washed after 1 h to remove oligodendrocytes progenitors and then treated with recombinant IL-4 (0.08, 1.24, 2.5, 5, 10, 20, and 40 ng/ml), (R&D system) for 12 h. Cultures were paraformaldehyde fixed for 5 min at room temperature and used for immunofluorescence. While, parallel cultures were used to obtain total RNA extracts and used for real time PCR.

Rossi C., Cusimano M., Zambito M., Finardi A., Capotondo A., Garcia-Manteiga J.M., Comi G., Furlan R., Martino G, & Muzio L. (2018). Interleukin 4 modulates microglia homeostasis and attenuates the early slowly progressive phase of amyotrophic lateral sclerosis. Cell Death & Disease, 9(2), 250.

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