Interleukin 4 (il 4)

Manufactured by STEMCELL

Sourced in Canada

IL-4 is a recombinant human cytokine that plays a key role in the regulation of immune responses. It is primarily produced by activated T cells and mast cells, and acts on a variety of target cells to modulate their function.

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Lab products found in correlation

Gm csf, by STEMCELL (3 mentions) Lipopolysaccharides (lps), by Merck Group (2 mentions) Gm csf, by Thermo Fisher Scientific (1 mentions) Sepmate, by STEMCELL (1 mentions) Lymphoprep, by STEMCELL (1 mentions) Stable glutamine, by Capricorn (1 mentions) Recombinant gm csf, by Miltenyi Biotec (1 mentions) Lipopolysaccharides (lps), by Nacalai Tesque (1 mentions) Penicillin streptomycin, by Nacalai Tesque (1 mentions) Mojosort magnetic cell separation system, by BioLegend (1 mentions) Lymphoprep, by Axis-Shield (1 mentions) Ifn γ, by Miltenyi Biotec (1 mentions) Fetal bovine serum (fbs), by Capricorn (1 mentions) Il 10, by STEMCELL (1 mentions) Blasticidin, by Thermo Fisher Scientific (1 mentions) Aim 5 media, by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Dmem f12, by Thermo Fisher Scientific (1 mentions) Gm csf, by R&D Systems (1 mentions) Fetal bovine serum (fbs), by Avantor (1 mentions)

9 protocols using interleukin 4 (il 4)

Dendritic Cell Differentiation and Maturation

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Monocyte isolation and DC differentiation were performed as described previously.⁴ (link) Briefly, monocytes were obtained using the adherence method, where PBMCs were plated in adherent plastic plates (Sarstedt, Nümbrecht, Germany) in medium (X-Vivo 15 medium, Lonza, Basel, Switzerland) supplemented with 5% human serum, 2 mM L-glutamine and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA), 1,000 U/mL (100 ng/mL) IL-4, and 800 U/mL (50 ng/mL) Granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from STEMCELL Technologies) and incubated in a CO₂, 37°C incubator for 7 days. On day 4, the medium was replaced with fresh medium supplemented with IL-4 and GM-CSF. On day 7, DCs were matured with maturation medium containing 1,000 U/mL (100 ng/mL) IL-4, 800 U/mL (50 ng/mL) GM-CSF, 10 ng/mL tumor necrosis factor alpha (TNF-α) (STEMCELL Technologies), 1 μg/mL PGE2 (Sigma), 10 ng/mL IL-1β (Feldan, Quebec City, QC, Canada), and 100 ng/mL IL-6 (Miltenyi Biotec, Bergisch Gladbach, Germany) and loaded with the desired peptide (1 μg/mL LMP2_426–434, [CLGGLLTMV] or 1 μg/mL WT1_37–45 [VLDFAPPGA], both from JPT Peptides (Berlin, Germany). Last, DC medium was supplemented with interferon γ (IFN-γ; 1,000U/mL, Feldan) for the last 24 h of maturation.

Lak S., Janelle V., Djedid A., Boudreau G., Brasey A., Lisi V., Smaani A., Carli C., Busque L., Lavallée V.P, & Delisle J.S. (2022). Combined PD-L1 and TIM3 blockade improves expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy. Molecular Therapy. Methods & Clinical Development, 27, 230-245.

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PBMC Isolation and Monocyte-Derived Dendritic Cell Generation

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Peripheral blood mononuclear cells (PBMCs) were isolated using lymphoprep and SepMate (StemCell Technologies). Monocytes were isolated from PBMCs using EasySep™ Mouse Monocyte Isolation Kit. 500,000 monocytes/ml were cultured in complete RPMI with FBS supplemented with 50 ng/ml GM-CSF (Peprotech) plus 25 ng/ml IL-4 (StemCell Technologies) in 24-well plates for 3 days.

Ebrahimi-Nik H., Corwin W.L., Shcheglova T., Das Mohapatra A., Mandoiu I.I, & Srivastava P.K. (2018). CD11c+ MHCIIlo GM-CSF-bone marrow-derived dendritic cells act as antigen donor cells and as antigen presenting cells in neoepitope-elicited tumor immunity against a mouse fibrosarcoma. Cancer Immunology, Immunotherapy, 67(9), 1449-1459.

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Isolation and Polarization of M1/M2 Macrophages

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Thirty ml of peripheral blood was withdrawn from three groups of CAD patients. Peripheral blood mononuclear cells (PBMCs) were isolated by collecting the buffy coat generated after Lymphoprep™ (Axis Shield, Oslo, Norway) density centrifugation. Mojosort™ magnetic cell separation system (Biolegend, San Diego, California) was employed to further isolate monocytes from the pooled PBMCs. Next, monocytes were re-suspended (1 × 10⁶/ml) in RPMI1640, with stable glutamine (Capricorn Scientific, Germany) and seeded into 25 cm² tissue culture flask (SPL Life Sciences, Korea). Monocytes were allowed to adhere at 37 °C, 5% CO₂ for 3 h. Non-adherent cells were washed off using RPMI 1640 with stable glutamine media. The adherent monocytes were cultured for 5 days in RPMI 1640 with stable glutamine media supplemented with 10% heat-inactivated fetal bovine serum (Capricorn Scientific, Germany), 1% penicillin-streptomycin (Nacalai Tesque, Japan), and 20 ng/ml recombinant GMCSF (Miltenyi Biotec, Germany) for M1 macrophage or 10 ng/ml recombinant MCSF (Gold Biotechnology, Missouri) to generate M2 macrophages. After day 5, M1 macrophages were polarized with 10 ng/ml LPS (Nacalai Tesque, Japan) and 20 ng/ml IFN-γ (Miltenyi Biotec, Germany) for 2 days. Meanwhile, 20 ng/ml IL-4 and 20 ng/ml IL-13 (Stemcell Technologies, Canada) were added into culture media to polarize M2 macrophages.

Mohd Idrus F.N., Ahmad N.S., Hoe C.H., Azlan M., Norfuad F.A., Yusof Z., Wan Isa W.Y., Mohamed Ali A.A, & Yvonne-Tee G.B. (2021). Differential polarization and the expression of efferocytosis receptor MerTK on M1 and M2 macrophages isolated from coronary artery disease patients. BMC Immunology, 22, 21.

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Polarizing and Conditioning RAW 264.7 Cells

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RAW 264.7 cells were harvested (by cell scraper) and seeded at 25 × 10⁵ cells/T25 flask. After 24h, cells were polarized in 5 ml serum free DMEM as follows: For M0 polarization, serum free media (SFM) alone was added. For M1 polarization, 20 ng/ml interferon gamma + 100ng/ml lipopolysaccharide (Stemcell Technologies, Vancouver) was added and for M2 polarization 20 ng/ml of IL-4, and 20 ng/ml IL-10 (Stemcell Technologies) was added. Cells were polarized for 24 h, thoroughly washed with PBS then 5 ml of fresh SFM was added. After another 24 hrs, the conditioned media (CM) was filtered (0.22 μm filter) and stored at −20°C until needed.

Hudson A.L., Parker N.R., Khong P., Parkinson J.F., Dwight T., Ikin R.J., Zhu Y., Chen J., Wheeler H.R, & Howell V.M. (2018). Glioblastoma Recurrence Correlates With Increased APE1 and Polarization Toward an Immuno-Suppressive Microenvironment. Frontiers in Oncology, 8, 314.

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Generation of Stable Cell Lines for Knockdown

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HeLa R19, A375, and Hs683 cells were maintained in high-glucose DMEM supplemented with 0.1 mM nonessential amino acids (Invitrogen) and 10% fetal bovine serum (FBS; Sigma). M059J cells were grown in DMEM:F12 (Invitrogen) supplemented with 0.05 mM nonessential amino acids and 10% FBS. To generate stable cell lines with dox-inducible knockdown of genes of interest, A375 cells were first transfected with BstZ17I-digested pcDNA6/TR (Invitrogen), the tetracycline repressor expression vector, and grown under blasticidin (5 μg/mL; Invitrogen) selection. blasticidin-resistant clones were screened for dox-inducible RLuc expression after transient transfection with pcDNA5/TO/FRT/Luc (38 (link)), and the clone with the lowest RLuc basal level:highest induction ratio was selected and expanded as a host cell line. Stable cell lines with dox-inducible knockdown of ADAR, DHX9, DHX58 (LGP2), or EIF2AK2 (PKR) were generated with shRNA-expressing vectors (see below); G418-resistant (600 μg/mL; Invitrogen) clones were screened by immunoblot of lysates from control/dox-induced cells (1 μg/mL; 48–72 h) for the highest level of target protein depletion. MdDCs were generated from leukopak-derived PBMCs (Stemcell Tech) by allowing monocytes to adhere (1 h) at 37°C in AIM-V media (Thermo Fisher), followed by 6 days of culture in 50 μg/mL GMCSF (R&D systems) and 20 ng/mL IL-4 (Stemcell Tech).

Dobrikov M.I., Dobrikova E.Y., McKay Z.P., Kastan J.P., Brown M.C, & Gromeier M. (2022). PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling. mBio, 13(3), e00854-22.

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Macrophage Differentiation from THP-1 Cells

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To induce macrophage differentiation, 4 × 10^e5 low-passage Thp1, a human monocytic leukemia cell line (CSL), were seeded onto 12-well plate in RPMI GlutaMAX (Invitrogen) supplemented with 10% FBS (VWR) and 1% penicillin/streptomycin (Sigma-Aldrich), and treated with 162 nmol/L Phorbol 12-Myristate 13-Acetate (Sigma-Aldrich) and 20 ng/mL IL4 (StemCell), according to the previously published protocol (40 (link)). Follistatin (FST, Peprotech) was used at 50 ng/mL. Thp1 cells were maintained at 37°C in humidified conditions with 5% CO₂ and were confirmed as negative for Mycoplasma prior to use in assays.

Pich-Bavastro C., Yerly L., Di Domizio J., Tissot-Renaud S., Gilliet M, & Kuonen F. (2023). Activin A–Mediated Polarization of Cancer-Associated Fibroblasts and Macrophages Confers Resistance to Checkpoint Immunotherapy in Skin Cancer. Clinical Cancer Research, 29(17), 3498-3513.

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Monocyte-Derived Dendritic Cell Differentiation and Maturation

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Isolated PBMCs were re-suspended in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and 2 mM GlutaMAX (Gibco) and seeded in 25-cm² cultural flasks (Corning) at a concentration of 6∗10⁶ cell/mL. After 2 h, the unbound cells were removed, and the media were changed to fresh, supplemented with growth factors—IL-4 (100 ng/mL) and GM-CSF (50 ng/mL) (STEMCELL Technologies), and then cultivated for 6 days with a change of half of the media volume every 2 days. After 6 days, the full medium volume was changed to a fresh portion with bacterial lipopolysaccharide (10 μg/mL) and cultivated for 24 h for DC maturation. MoDCs were analyzed for the expression of CD11c, CD80, and HLA-DR on the membrane surface using the NovoCyte flow cytometer (ACEA Biosciences).

Ishina I.A., Kurbatskaia I.N., Mamedov A.E., Shramova E.I., Deyev S.M., Nurbaeva K.S., Rubtsov Y.P., Belogurov AA J.r., Gabibov A.G, & Zakharova M.Y. (2024). Genetically engineered CD80–pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement. Frontiers in Bioengineering and Biotechnology, 11, 1341685.

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Generating Dendritic Cells from Bone Marrow

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Dendritic cells were generated from bone marrow isolated from femurs and tibia from indicated mice. 2×10⁶ bone marrow cells were cultured in media containing GM-CSF (25 ng/ml; Stem Cell Technologies, Vancouver, Canada) and IL-4 (5 ng/ml; Stem Cell Technologies) for 7 days[18 (link)]. On day 3, cultures were replenished with media containing GM-CSF and IL-4. On Day 6, immature DCs were stimulated with LPS (1μg/ml; Sigma-Aldrich, St. Louis, MO, USA) for 24 hrs. DCs were cultured at 37°C, 5% CO₂ in RPMI 1640 (Invitrogen, Waltham, MA, USA) supplemented with 10% FBS, 100IU/mL penicillin, 100 μg/ml streptomycin, 50uM β-mercaptoethanol, and 2mM L-glutamine.

Amir M., Campbell S., Kamenecka T.M, & Solt L.A. (2020). Pharmacological modulation and genetic deletion of REV-ERBα and REV-ERBβ regulates dendritic cell development. Biochemical and biophysical research communications, 527(4), 1000-1007.

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Human Monocyte Differentiation to M1 and M2 Macrophages

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Fresh blood samples from healthy donors were collected, and PBMCs were isolated by density gradient centrifugation using SepMate-50 tubes (STEMCELL Technologies). Isolation of monocytes was performed using the classical monocyte isolation kit (Miltenyi Biotec) according to the manufacture’s protocol. After isolation of CD14⁺ monocytes, cells were cultured in ImmunoCult^™-SF Macrophage Medium (STEMCELL Technologies) along with 5 μg/mL recombinant human recombinant macrophage colony stimulating factor (M-CSF, STEMCELL Technologies). Monocytes were then cultured at 37°C in a humidified incubator with 5% CO₂ until day 4. For M1 differentiation, 10 ng/mL LPS (MilliporeSigma) and 50 ng/mL IFNγ (STEMCELL Technologies) were added to the culture. For M2 differentiation, 10 ng/mL IL4 (STEMCELL Technologies) was added to the culture according to the manufacture’s protocol. Macrophages were harvested on day 6 and used in the following assays as indicated.

Sakemura R.L., Hefazi M., Cox M.J., Siegler E.L., Sinha S., Hansen M.J., Stewart C.M., Feigin J.M., Roman C.M., Schick K.J., Can I., Tapper E.E., Horvei P., Adada M.M., Bezerra E.D., Fonkoua L.A., Ruff M.W., Forsman C.L., Nevala W.K., Boysen J.C., Tschumper R.C., Grand C.L., Kuchimanchi K.R., Mouritsen L., Foulks J.M., Warner S.L., Call T.G., Parikh S.A., Ding W., Kay N.E, & Kenderian S.S. (2023). AXL inhibition improves the anti-tumor activity of chimeric antigen receptor T cells. Cancer immunology research, 11(9), 1222-1236.

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