IL-2RβTg mice were i.p. injected with 1 µg of recombinant IL-4 (Peprotech, Cranbury, NJ, USA) complexed with anti-IL-4 antibodies (eBioscience) or vehicle every other day for 2 weeks. An IL-4 and anti-IL-4 antibody complex was prepared as previously described [19 (link)] with slight modifications. Then, 20 µg of recombinant IL-4 (Peprotech) powder was reconstituted with 80 µL distilled water and mixed with 120 µL anti-IL-4 antibody (1 mg/mL; 11B11; eBioscience). The mixture was incubated for 10 min at room temperature, diluted 10-fold with PBS, and kept at 4 °C until further use.
Recombinant il 4
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada, Japan, United Kingdom, Germany
Recombinant IL-4 is a cytokine produced using recombinant DNA technology. It functions as an important regulator of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (6 mentions)
Gm csf, by Thermo Fisher Scientific (4 mentions)
Anti il 4 antibody, by Thermo Fisher Scientific (3 mentions)
Il 13, by Thermo Fisher Scientific (3 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Aim 5 medium, by Thermo Fisher Scientific (2 mentions)
Recombinant il 2, by Thermo Fisher Scientific (2 mentions)
Anti interferon γ monoclonal antibody r4 6a2, by Thermo Fisher Scientific (2 mentions)
Recombinant ifn γ, by Thermo Fisher Scientific (2 mentions)
Il 12, by Thermo Fisher Scientific (2 mentions)
Tgf β, by R&D Systems (2 mentions)
Recombinant il 6, by R&D Systems (2 mentions)
Murine m csf, by Thermo Fisher Scientific (1 mentions)
Gm csf, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Recombinant gm csf, by Thermo Fisher Scientific (1 mentions)
38 protocols using recombinant il 4
1
IL-4 Complexation and Administration
2
Isolation and Alternative Activation of BMDMs
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Bone marrow-derived macrophages (BMDMs) were isolated and cultured as described previously15 (link). Briefly, bone marrow cells from wild type C57BL/6 mice were isolated from the tibias and femurs and treated with 20 ng/ml of recombinant mouse macrophage colony stimulating factor (Murine M-CSF, PeproTech Canada) for 7 days. After 7 days, bone marrow derived macrophages were treated for either 18 or 30 hours with recombinant IL-4 (20 ng/ml), IL-13 (50 ng/ml), alone, or in combination with OSM (50 ng/ml) or IL-6 (50 ng/ml) (PeproTech Canada). Alternative activation of macrophages was assessed in the cell lysate by measuring arginase-1 protein by western blotting and by arginase-1 and CD206 by flow cytometry. In some instances, BMDMs were lysed and RNA was isolated for NanoString gene expression analysis.
3
Anti-IL-4 mAb Neutralization Efficacy
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Neutralizing anti-IL-4 mAb was purified from culture supernatants of hybridoma (clone 11B11) using a protein G column kit (Kierkegaard & Perry Laboratories), and control rat IgG was purchased from ICN Pharmaceuticals, Inc. (Aurora, OH, USA). Mice were injected intraperitoneally with either Ab at 200 μg/mouse one day before and on day 0, 3, and 7 after infection. Anti-IL-4 mAb treatment reduced the level of IL-4 by more than 90% in the infected lungs compared to that in the control rat IgG-treated mice. In addition, in an in vitro experiment, 10 μg/ml anti-IL-4 mAb showed an approximately 50% neutralizing effect on the suppression of IL-12p40 synthesis by bone marrow-derived dendritic cells (BM-DCs) stimulated with phosphorothioated CpG1826 (100 ng/ml: synthesized by Hokkaido System Science [Sapporo, Japan]) caused by recombinant IL-4 (100 ng/ml: PeproTech, Rocky Hill, NJ, USA), and 100 μg/ml anti-IL-4 mAb completely abrogated this suppression. BM-DCs were prepared by culturing bone marrow cells from C57BL/6 mice with 20 ng/ml murine granulocyte-macrophage colony-stimulating factor (GM-CSF, Wako, Osaka, Japan) for 8 days.
4
Murine Dendritic Cell Culture from Bone Marrow
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Rats were killed by CO2 asphyxia. Femur and tibia bones from arthritic (CIA day28) and age matched controls were aseptically dissected. Under sterile conditions whole bone marrow was flushed out with RPMI-1640 Medium (R7509, Sigma Aldrich) supplemented with 2 mM glutamine, 1%penicillin/streptomycin solution, 10% fetal bovine serum, 10 mM HEPES, and 50 µM 2-mercaptoethanol (all from Sigma Aldrich), and collected after passing a 70 µm mesh cell strainer. After erythrocyte lysis (Qiagen), cell number and viability was checked by trypan blue exclusion. 1.5 × 106 cells per well were seeded in 6-well plates (3516, Corning) in medium (see above) supplemented with 5 ng/ml recombinant GM-CSF, 5 ng/ml recombinant IL-4 and 25 ng/ml mFLT3-ligand (all from Peprotech, Rocky Hill, NJ, USA). Medium was changed on day 3 and 6 after seeding. On day 10, cells were carefully detached by pipetting, and passaged onto new plates. Medium was changed again on day 12, supplemented with 2.5 ng/ml GM-CSF only. On day 14 loose cell-clusters with dendritic shaped cells were collected by pipetting, and subsequently used for co-culture or in vivo experiments. The method was adapted and slightly modified from various protocols29 (link)–32 (link).
5
Monocyte-Derived Dendritic Cell Differentiation
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Monocyte derived dendritic cells (moDC) were differentiated from CD14+ peripheral blood monocytes obtained from the same skin donors. Briefly, 20 mL of total blood were obtained from each donor, and PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque (Histopaque 1077, Sigma Aldrich Chemical Co., St. Louis, MO); CD14+ monocytes were positively selected from PBMCs using anti-CD14 microbeads (Cat. 130-050-201) (Miltenyi Biotech, Auburn, CA) and a magnetic cell separator (MACS) (Miltenyi Biotech, Auburn, CA) according to manufacturer’s instructions. Monocytes from different donors were seeded in plastic flasks at a density of 1x106 per ml of differentiation monocyte culture media (RPMI supplemented with 10% FBS, sodium pyruvate, L-Glutamine, NEAA, vitamins, recombinant human GM-CSF ([50ng/mL, Peprotech, London, United Kingdom] and recombinant IL-4 [30 ng/mL, Peprotech, London, United Kingdom]) in a CO2 atmosphere at 37°C. Cytokines were replenished every two days and cells were used on day 6 as immature DC. The appropriate phenotype of immature DC was confirmed by flow cytometry prior to each experiment (all DCs were CD14-, CD1a+, HLA-DR+, CD83-, CD86+).
6
Allergen-specific Th2 Cell Preparation
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Allergen-specific Th2 cells were prepared as described previously [16 (link)18 (link)]. Briefly, OVA-specific naïve CD4+ T cells were isolated from splenocytes of DO11.10/RAG-2-/- mice by positive selection using CD4 microbeads and a magnetic-activated cell sorting system (Miltenyi, Bergisch Gladbach, Germany). Cells were cultured with X-ray-irradiated splenocytes in DMEM-nutrient mixture F12-HAM medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum. At the start of culture, 0.3-μM synthetic OVA323-339 peptide (Scrum Inc., Tokyo, Japan), 10-U/mL recombinant IL-2 (Shionogi, Osaka, Japan), 10-U/mL recombinant IL-4 (PeproTech, Rocky Hill, NJ, USA), and 10-μg/mL anti-IFN-γ monoclonal antibody (R4-6A2, eBioscience, San Diego, CA, USA) were added. Seven days after the stimulation, cells were harvested and used for the adoptive transfer and in vitro experiments.
7
Isolation and Culture of Dendritic Cells
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Peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated by leukapheresis. Leukapheresis was approved by the Ethics committee of the University Hospital Regensburg (Ethic Vote July 2010 #09/066b and #09/066c); all human participants gave written informed consent. Human monocytes were isolated from PBMCs by density gradient centrifugation over Ficoll/Hypaque followed by counterflow centrifugation elutriation [41 (link)]. Immature dendritic cells (iDC) were generated from human blood monoctes. Monocytes were cultured in culture flasks at a concentration of 1 × 106 cells/1.5 mL in RPMI 1640 (Gibco, Waltham, MA, USA) for seven days. The medium was supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine (Biochrom, Berlin, Germany), 50 U/mL penicillin (Gibco, Waltham, MA, USA), 50 U/mL of streptomycin (Gibco), 225 U/mL granulocyte macrophage colony stimulating factor (GMCSF, Peprotech, Hamburg, Germany), and 144 U/mL recombinant IL-4 (Peprotech). Cell number, cell size, and cell viability were determined using the CASY cell analyzer system (Casy® Modell TT, OLS Omni Life Science, Bremen, Germany). Appropriate cursor settings for determining cell number and viability were established for each cell type.
8
Macrophage Depletion and Manipulation for Wound Healing
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For macrophage depletion, 50 μg of clodronate liposomes (Clod-Lipo) or control liposomes (PBS-Lipo) (Encapsula NanoSciences, CLD-8901) were injected intraperitoneally 2 days before surgery. In order to determine the phagocytosis ability of peritoneal cavity cells, 50 μg of Fluoroliposome-DiD (Encapsula Nano Sciences, CLD-8913) were injected intraperitoneally 2 days after surgery. For blocking macrophage-fibrin interaction, 50 μg anti-CD11b neutralizing antibody (clone: 5C6) (Invitrogen, MA5-16528) or rat IgG2b control antibody (clone: eB149/10H5; eBioscience,16–4031–85) was injected intraperitoneally 3 h before and immediately after surgery. For increasing macrophages, IL-4c (5 μg recombinant IL-4 (PeproTech, 214–14) and 25 μg stabilizing monoclonal anti-IL-4 antibody (clone: BVD4-1D11; BD Biosciences, 554387)) dissolved in 100 μl PBS were intraperitoneally injected to normal mice or administrated into the peritoneal cavity immediately after the ischemic button creation. An equivalent volume of PBS was injected as a control.
9
Signaling Pathway Reagents and Antibodies
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Recombinant IL6 was purchased from R&D Technology. PD98059 was from Sigma-Aldrich. Recombinant IL4 and IL13 were purchased from Peprotech. SP600125 and SB203580 were obtained from Selleck Chemicals. The following primary antibodies to p-ERK (Thr202/Tyr204), ERK, p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), p38 were from Cell Signaling.
10
Murine Dendritic Cell-T Cell Co-culture
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Mouse bone marrow cells were isolated from the tibiae and femurs and treated with red blood cell lysing buffer. Cells were cultured with phenol red-free RPMI 1640 medium (HyClone, Shanghai, China) supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 μg/ml penicillin-streptomycin (Sigma, St Louis, MO, USA), 10 ng/ml recombinant granulocyte‐macrophage CSF (PeproTech, Rocky Hill, NJ, USA), and 1 ng/ml recombinant IL‐4 (PeproTech, Rocky Hill, NJ, USA) at 37°C with 5% CO2. Non-adherent and loosely adherent cells were harvested after cultured for 6 d. Cells with specific DC markers were isolated by fluorescence‐activated cell sorting. Then mouse bone marrow-derived dendritic cells (BMDCs) were cultured for experiments. CD4+ T lymphocytes were isolated by cell negative isolation kit (Invitrogen Dynal AS, Oslo, Norway) and cultured in RPMI-1640 medium (HyClone, Shanghai, China) supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 μg/ml penicillin-streptomycin (Sigma, St Louis, MO, USA), 1% non-essential Aas, 4 mM l -glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and 2 × 10−5 M 2-β-mercaptoethanol at 37°C with 5% CO2. BMDCs were stimulated by LPS (100 ng/ml; Sigma-Aldrich, Missouri, USA) for 24 h before co-culture with CD4+ T lymphocytes. T lymphocytes were cultured with DCs at a ratio of 5:1 and the cells were analyzed after 3 d.
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