LPS (Escherichia coli serotype O55:B5) and adenosine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deuterated adenosine was from CDN Isotopes (Quebec, Canada). adenosine 5′-triphosphate disodium salt (ATP) was from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human M-CSF, IFNγ, and IL-10 were obtained from Peprotech (Rocky Hill, NJ, USA). Recombinant human GM-CSF and IL-4 were from Novartis AG (Basel, Switzerland). The RPMI 1640 medium, l -glutamine, streptomycin, penicillin, and heat-inactivated fetal calf serum (FCS) were obtained from Gibco, Thermo Fisher Scientific. CD39 inhibitor POM-1 was from Tocris Bioscience (Bristol, UK). The cell proliferation dye CFSE and calcium sensor Fluo-4, AM was from Molecular Probes, Thermo Fisher Scientific. Brilliant Violet 421-conjugated streptavidin used as a second step in flow cytometry analyses was purchased from BioLegend (San Diego, CA, USA). Phorbol 12-myristate 13-acetate (PMA), ionomycin calcium salt (ionomycin) from S. conglobatus and monensin A sodium salt (monensin) were purchased from Sigma-Aldrich.
Interleukin 4 (il 4)
Manufactured by Novartis
Sourced in Switzerland
IL-4 is a lab equipment product used for the detection and quantification of the cytokine interleukin-4 in various biological samples. It functions as an analytical tool for researchers studying immune system regulation and related fields.
Automatically generated - may contain errors
Lab products found in correlation
Ifn γ, by Thermo Fisher Scientific (3 mentions)
Gm csf, by Novartis (3 mentions)
M csf, by Novartis (3 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (3 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Recombinant human gm csf, by Novartis (2 mentions)
M csf, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Gibco tryple select 10, by Thermo Fisher Scientific (2 mentions)
Rpmi medium, by Merck Group (2 mentions)
Adenosine, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Brilliant violet 421 conjugated streptavidin, by BioLegend (1 mentions)
Adenosine 5 triphosphate disodium salt atp, by Thermo Fisher Scientific (1 mentions)
Cd39 inhibitor pom1, by Bio-Techne (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using interleukin 4 (il 4)
1
Macrophage Activation Assay Protocol
2
Monocyte Differentiation Markers
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LPS (Escherichia coli serotype O55:B5) was purchased from Sigma-Aldrich (Munich, Germany). Recombinant human M-CSF, IFNγ and IL-10 were obtained from Peprotech (Rocky Hill, NJ, USA). Recombinant human GM-CSF and IL-4 were from Novartis AG (Basel, Switzerland). The HLDA10 mAb panel, tested within the 10th Human Leukocyte Differentiation Antigen Workshop, in Wollongong, Australia, is detailed at http://www.hcdm.org . Alexa Fluor 647-conjugated monomeric streptavidin used at a final concentration of 2.5 μg ml−1 was prepared in-house. Allophycocyanin-conjugated goat anti-mouse IgG+IgM Ab used at a final dilution of 1:2500 was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Control antibodies included: Pacific Blue-, FITC- or PE-labelled mouse IgG1 isotype control mAb, clone MOPC-21 (Biolegend, San Diego, CA, USA), Pacific Blue-conjugated CD14 mAb, clone MEM-18, unlabelled mouse IgG1 isotype control mAb, clone PPV06 (both from EXBIO, Prague, Czech Republic) and biotin-labelled mouse IgG1 isotype control mAb MCA928B (AbD Serotec, Oxford, UK).
3
Macrophage Differentiation and Activation
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CD14+ monocytes were differentiated by seeding the cells at a density of 7.7 × 105 cells per well into 12-well plates in RPMI complete medium supplemented with either 50 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF (PeproTech) for 7 days to obtain M1-like pro-inflammatory or M2-like anti-inflammatory macrophages, respectively. The differentiated macrophages were further activated with 100 ng/ml LPS (Sigma) plus 25 ng/ml IFN-γ (PeproTech) or 100 ng/ml IL-4 (Novartis) for 2 days in the presence of either 10 ng/ml GM-CSF or M-CSF. The accumulation of VLCFAs in blood monocytes and in vitro differentiated macrophages was analysed by using GC–MS as described (Weber et al., 2014 (link)).
4
Differentiation of M1 and M2 Macrophages
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Dendritic cells were obtained by culturing 1 × 106 CD14+ monocytes as previously described 33 . To obtain the herein termed M1 and M2 macrophage‐like cells, 0.5 × 106 CD14+ monocytes were cultured for 4 days with 5 ng/ml human recombinant Granulocyte macrophage‐colony stimulating (GM‐CSF) (Miltenyi Biotec) or 20 ng/ml M‐CSF (Miltenyi Biotec) respectively. M1 were then treated with 20 ng/ml IFN‐γ (BD Biosciences) and 100 ng/ml Lipopolysaccharide (LPS) (Sigma‐Aldrich), whereas M2 with 20 ng/ml IL‐4 (by Dr. Schweighoffer, Novartis, Vienna, Austria) and 100 ng/ml LPS (Sigma‐Aldrich), for 2 days. All cultures were performed in 24‐well plates (Corning, Corning, NY, USA) at 37°C in 1 ml of Roswell Park Memorial Institute (RPMI) complete medium.
5
Generation of Immature and Tolerogenic DCs
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Immature DCs were generated from buffy coats from healthy, anonymous donors (provided by the blood bank Salzburg, Austria) as described previously43 (link). Briefly, density gradient centrifugation using Histopaque-1077 (Sigma) was performed to isolate PBMCs. ACK buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA) was used to lyse erythrocytes, and monocyte adherence was performed for 75 min at 37 °C and 5% CO2 in DC medium [RPMI 1640 (Sigma), 10% foetal calf serum (PAA), 2 mM l-glutamine, 100 U/ml penicillin/streptomycin (Sigma), 50 μM β-mercaptoethanol (Gibco Laboratories)]. After washing, adherent monocytes were cultured in DC medium supplemented with 50 ng/ml GM-CSF and 50 ng/ml IL-4 (generous gift from Novartis) for 7 days to generate immature iDCs or with additional 30 ng/ml IL-10 (R&D Systems) to obtain tolerogenic DCs. On day 3 of differentiation, fresh supplemented medium was added to the cells.
6
Isolation and Stimulation of PBMCs
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Human PBMCs were isolated from blood donations of healthy, anonymous donors (kindly provided by the blood bank Salzburg, Austria) using density gradient centrifugation.23 0.5 × 106 cells/mL PBMCs were seeded in IMDM (Sigma) supplemented with 5% FCS (PAA), 100 U/mL penicillin (Sigma), and 100 µg/mL streptomycin (Sigma). After 7 days of stimulation with 10 µg/mL of OVA, nOVA, nOVAmax (LPS concentration adjusted), or the respective amount of LPS, cells, and supernatants were harvested for flow cytometry and ELISA, respectively.
For the isolation of monocytes, PBMCs were allowed to adhere for 70 minutes. After removal of nonadherent cells, monocytes were differentiated to moDCs in the presence of 50 ng/mL GM‐CSF and 50 ng/mL IL‐4 (Novartis) for 6 days. There upon 1 × 105 moDCs/mL was seeded and either left untreated or stimulated with OVA, nOVA, nOVAmax (LPS concentration not adjusted), and LPS for indicated times.
For the isolation of monocytes, PBMCs were allowed to adhere for 70 minutes. After removal of nonadherent cells, monocytes were differentiated to moDCs in the presence of 50 ng/mL GM‐CSF and 50 ng/mL IL‐4 (Novartis) for 6 days. There upon 1 × 105 moDCs/mL was seeded and either left untreated or stimulated with OVA, nOVA, nOVAmax (LPS concentration not adjusted), and LPS for indicated times.
7
Cytokine-Stimulated Airway Epithelial Cell Coculture
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ALI cultures were stimulated with different cytokines to the basolateral compartment in the above-mentioned BEGM. IL-4 (Novartis, Basel, Switzerland), IL-5 (PeproTech, Rocky Hill, NJ), IL-13 (eBioscience, San Diego, Calif), IL-25, IL-33, and TSLP (R&D Systems, Minneapolis, Minn) were all used at concentrations of 10 and 50 ng/mL. For the blocking experiments, we used anti-IL-4 (2 mg/mL) and anti-IL-13 (3 mg/mL) antibodies (both from R&D Systems) at the optimum dose, according to the manufacturer's instructions. These doses were sufficient to neutralize the transepithelial electrical resistance (TER)-decreasing effect of exogenously added IL-4 and IL-13 in ALI cultures of control HBECs. T H 2 cells were in vitro differentiated from naive T cells into T H 2 cells, as described previously. 29, 30 BEGM was mixed 1:1 with complete RPMI-1640 (Sigma-Aldrich) containing 10% FCS and antibiotics for cocultured HBECs and T H 2 cells. HBECs were cocultured with T H 2 cells at a concentration of 1 3 10 5 and 5 3 10 5 cells/well added to the basolateral compartment in the same total volume of 0.6 mL. T H 2 cells were stimulated with anti-CD28 (125 ng/mL), anti-CD3 (125 ng/mL), anti-CD2 (125 ng/mL), and IL-2 (100 U/mL). As a control, epithelial cells alone were cultured by using the same conditions.
8
Macrophage Differentiation and Polarization Protocol
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For macrophage differentiation and polarization, CD14+ monocytes (1 × 106 cells/well) were isolated and characterized by flow cytometry as described previously15 , 16 and seeded in RPMI medium (Sigma‐Aldrich) containing 1% Penicillin/Streptomycin, 1% glutamine, 1% Fungizone, and 10% fetal calf serum, supplemented with either 50 ng/mL human recombinant GM‐CSF or M‐CSF (PeproTech) for 7 days. For β‐oxidation assays, M‐CSF‐differentiated macrophages were polarized into an anti‐inflammatory phenotype with 100 ng/ml IL‐4 (Novartis) and 10 ng/ml M‐CSF for 2 days. To analyse pro‐inflammatory cytokine gene expression, M‐CSF‐differentiated macrophages were stimulated with 100 ng/ml LPS (E. coli 055:B5, #L4005, Sigma) and treated with vehicle control or Vorinostat (#10009929, Cayman Chemicals) for 24 h. ELISA to measure secreted IL12p40 was described previously.16 To determine mean cell size and number of adherent or nonadherent cells, a CASY automated cell counter (Omni Life Sciences) was used. For detachment, adherent macrophages were incubated with 300 µL Gibco™ TrypLE™ Select (10×) (Gibco, Life Technologies) for 15 min at 37°C shaking every few minutes and was gently removed with a cell scraper.
9
Macrophage Polarization and Cytokine Expression
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CD14+ monocytes (1 × 106 cells/well) were cultured in RPMI medium (Sigma Aldrich) containing 1% Penicillin/Streptomycin, 1% glutamine, 1% Fungizone and 10% FCS, supplemented with 50 ng/mL human recombinant M‐CSF (PeproTech) for 7 days. For use in β‐oxidation assays, M‐CSF‐differentiated macrophages were polarized with 100 ng/mL IL‐4 (Novartis) for 2 days in the presence of 10 ng/mL M‐CSF. The HDAC inhibitors SAHA (Cat.No.10009929, Cayman Chemicals) and MS‐275 (Cat.No.T6233, Target Mol) were added during the last 48 h. For analysis of pro‐inflammatory cytokine gene expression, M‐CSF‐differentiated macrophages were stimulated with 100 ng/mL LPS (E. coli 055:B5, Cat.no. L4005, Sigma) and treated with DMSO, MS‐275, or SAHA in concentrations as indicated for 24 h. For detachment, adherent macrophages were washed with PBS and incubated with 300 µL Gibco™TrypLE™Select(10×) (Gibco, Life Technologies) for 15 min at 37°C, and collected with a cell scraper after adding 300 µL PBS.
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