Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy human donors by standard density-gradient centrifugation with Ficoll-Paque (Amersham Biosciences). CD14+ cells were purified from PBMCs by high-gradient magnetic sorting, using the VarioMACS technique with anti-CD14 microbeads (Miltenyi Biotec GmbH). Cells were then cultured in complete RPMI 1640 medium (Life Technologies) supplemented with 10 % (v/v) fetal calf serum (FCS), 20 ng/ml human GM-CSF (R&D Systems) and 10 ng/ml human IL-4 (R&D Systems) for 6 days (immature DC) (Chen et al., 2008 (link)). Human whole blood was obtained from healthy donors at the Taipei Blood Center of the Taiwan Blood Services Foundation, under a protocol (AS-IRB02–103202) approved by the IRB of the Clinical Center of the Department of Health, Taiwan. Written informed consent was obtained from all donors. For mouse bone marrow-derived DC (BMDC), bone marrow cells were isolated from femurs and tibias and cultured in RPMI 1640 complete medium supplemented with 10% (v/v) FCS, L-Glutamine, pen/strep and 40 ng/ml recombinant mouse GM-CSF (R&D Systems) for 9 days (Chen et al., 2008 (link)).
Human il 4
Manufactured by R&D Systems
Sourced in United States
Human IL-4 is a recombinant human cytokine. It is a secreted protein that plays a central role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Human gm csf, by R&D Systems (3 mentions)
Anti cd14 microbeads, by Miltenyi Biotec (2 mentions)
Recombinant human m csf, by R&D Systems (2 mentions)
Human ifn γ, by R&D Systems (2 mentions)
Lipopolysaccharide lps, by Merck Group (2 mentions)
Recombinant human il 9, by R&D Systems (2 mentions)
Imdm medium, by Thermo Fisher Scientific (2 mentions)
Human il 2, by R&D Systems (2 mentions)
Cpg odn 2006, by InvivoGen (2 mentions)
Human recombinant hcd40l, by R&D Systems (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse gm csf, by R&D Systems (1 mentions)
Ficoll paque, by Cytiva (1 mentions)
Rpmi 1640 complete medium, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Variomacs technique, by Miltenyi Biotec (1 mentions)
Phorbol 12, by Merck Group (1 mentions)
Mouse xl cytokine array kit, by R&D Systems (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
20 protocols using human il 4
1
Isolation and differentiation of human and mouse dendritic cells
2
Macrophage Polarization from PBMCs
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Monocytes were purified from PBMCs using human CD14 MicroBeads (MACS) and then differentiated into macrophages in complete RPMI medium containing 20 ng/ml recombinant human M-CSF (R&D Systems) for 8 days. Human macrophages were harvested on day 8, and stimulated with 20 ng/ml human IL-4 (R&D Systems) or 50 ng/ml human IFN-γ (R&D Systems) plus 20 ng/ml LPS for 24 h.
3
Macrophage Polarization and Cytokine Profiling
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The RAW264.7 and U937 cells were seeded at 2 × 104 cells/well and cultured for 24 h. The U937 cells were differentiated into M0 macrophages with 20 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich). Both the RAW264.7 and U937 cells were either polarized into M1 or M2 macrophage using 100 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich) or 20 ng/ml human IL-4 (R&D System) respectively, with or without 0.1 μg/ml HKUOT-S2 protein. The non-polarized cells were used as controls. The U937 cell-derived M1 and M2 macrophage condition media (CM) were collected for cytokine array analysis using the Mouse XL Cytokine Array Kit (R&D Systems) according to the manufacturer's protocol.
4
Th9 Differentiation of CD4+ T Cells
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Naive CD4+ T cells were isolated and cocultured with vehicle-Exo, PA-Exo, NCI-Exo, and miR-107I-Exo, respectively. Th9 differentiation was induced with human IL-4 (10 ng/mL, R&D Systems), human TGF-β (1 ng/mL, R&D Systems), and anti-IFN-γ monoclonal antibodies (10 μg/mL, eBioscience) for 3 days of culture.
5
Immune Checkpoint Modulation Protocols
6
Quantifying Serum Cytokine Levels
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Serum level of IL-33 was determined with Human IL-33 Quantikine ELISA Kit (R&D Systems). IL-4, IL-6 and IL-13 levels in culture supernatants were also detected with Human IL-4, IL-6 and IL-13 Quantikine ELISA Kits (R&D Systems) respectively according to the manual’s instructions.
7
Keratinocyte Differentiation and Cytokine Regulation
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Human keratinocytes were isolated from neonatal foreskin as previously described [35 (link)]. PHK were cultured in Keratinocyte-SFM (Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). To differentiate PHK, cells were grown in DMEM (11965092, Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B. For cytokine experiments, the following reagents were added alone or in combination to the culture media from the time of differentiation and replaced with every 48-h media change: human IL-4 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-13 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-17A (1–100 ng/mL; R&D system), JAK inhibitor I (10 μM; Calbiochem, San Diego, CA, USA), and PD98056 (10 μM; Calbiochem, San Diego, CA, USA). For the epidermal organotypic model experiment, keratinocytes were grown as previously described [36 (link)]. For all cytokine treatment studies, keratinocytes were starved of growth factors for 24 h before stimulation with the indicated cytokines.
8
Cytokine Response to IL-4 and IL-9 in PBMCs
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PBMCs were incubated with recombinant human IL-9 (R&D systems, Minneapolis, MN); human IL-4 (E. coli derived, R&D systems, Minneapolis, MN) at a concentration of 10, 20, or 100 ng/ml; or combinations of IL-4 and IL-9 during the stimulation with metal nickel chloride (10 μg/ml) or medium, and then incubated at 37°C for 96 hours. Supernatants were collected for in vitro cytokine analysis of IFN-γ, IL-4 and IL-9.
9
Immune Modulation with Poly I:C and Cytokines
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Polyinosinic–polycytidylic acid sodium salt (Poly I:C) was purchased from Sigma-Aldrich, and recombinant human interleukin (IL)-1α and recombinant human IL-4 were purchased from PeproTech (Rocky Hill, NJ, USA). For western blot analysis, IL-4, IL-1β, IL-6, filaggrin (FLG), and loricrin (LOR) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human IL-4, IL-13, and enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (Minneapolis, MN, USA).
10
STAT6-Mediated Transcriptional Regulation
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HEK293T cells were cultured to confluence in a 96-well plate and transfected with 40 ng human STAT6, 80 ng luciferase reporter, 3.2 ng ß-galactosidase, and either the FLM-PC1, PC1-p30 or PC1-p15 plasmid. Four h after transfection, 1 ng/ml human IL-4 (R&D Systems) and culture media without penicillin and streptomycin were added to the cells. EGFP in pCDNA4/TO was used as a negative control, and the backbone vector was used for balancing the plasmid amounts in all transfections. Luciferase assays were carried out after 20 h of treatment with IL-4 with luciferase substrate (Promega), and ß-gal activity was detected using 2-nitrophenyl β-D-galactopyranoside in sodium phosphate buffer.
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