Recombinant murine FLT3L was purchased from eBioscience (San Diego, CA). Recombinant murine GM-CSF, murine IL4, murine and human IL17A was purchased from R&D Systems (Minneapolis, MN). TLR4 ligand lipopolysaccharide LPS and TLR3 ligand poly (I:C) were purchased from InvivoGen (San Diego, CA).
Murine il 4
Manufactured by R&D Systems
Sourced in Macao, United States, United Kingdom
Murine IL-4 is a recombinant mouse cytokine that belongs to the Interleukin-4 protein family. It is a soluble protein that plays a key role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Murine gm csf, by R&D Systems (4 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Recombinant murine gm csf, by R&D Systems (1 mentions)
Tlr3 ligand poly 1 c, by InvivoGen (1 mentions)
Human il 17a, by R&D Systems (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
C di amp, by InvivoGen (1 mentions)
2 3 cgamp, by InvivoGen (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
Proleukin, by Novartis (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Immunotools (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Hla dr antibody, by BD (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
1 2 distearoyl sn glycero 3 phosphocholine dspc, by Avanti Polar Lipids (1 mentions)
10 protocols using murine il 4
1
Recombinant Cytokine Stimulation Assay
2
BV2 Cell Stimulation with IL-4
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BV2 cells were obtained from Dr. Linda Van Eldik, University of Kentucky, Lexington, KY. Cells were grown on six-well plates in FX12 media containing 10% fetal bovine serum, 1% serum L-glutamate and 1% streptomycin (Life Technologies, Carlsbad, CA) until 80% confluency was obtained approximately 3 days after passage. Cells were starved of serum for 24 h prior to treatment. Cells were stimulated with an exogenous application of murine IL-4 (20 ng/ml; R & D Systems, Minneapolis, MN) in the serum-free medium added to the culture. Serum-free medium was applied as the no treatment control. Cells were incubated (5% CO2, 37°C) for 2, 4, 6, 8, 10, 16 and 24 h before removal. Media were aspirated, and cells were rinsed twice in Dulbecco’s phosphate-buffered saline pH 7.4 (DPBS) (Life Technologies, Carlsbad, CA) at a 1× dilution for 5 min and frozen at −80°C. Cell culture experiments were performed in triplicate and repeated at least three times at different passage numbers.
3
Macrophage Polarization and Cyclic Dinucleotide Treatments
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Using aseptic technique, femurs were harvested, and the bone marrow flushed from C57BL/6 and Tmem173gt mice for macrophage polarization studies. The BMDMs were expanded in culture for 7 d in the presence of L-929 mouse fibroblast conditioned medium as a source of M-CSF. Cells were re-seeded onto 6-well plates at 2 × 106 cells/well and polarized for 48 hrs with the addition of 50 ng/ml LPS (List Biological Labs) and 50 ng/ml murine IFNγ (Cedarlane) for M1, 40 ng/ml murine IL-4 (R&D Systems) for M2 or left untreated for M0 cells. These cells were subsequently treated with either 20 μg/ml DMXAA (Cedarlane), 20 or 40 μg/ml 2′3′-cGAMP or 20 μg/ml c-di-AMP (InvivoGen, San Diego, CA) for an additional 6 hrs in the absence of polarizing cytokines. cDN treatments were given in the presence of Lipofectamine-2000 (LF2000, Invitrogen) to allow for entry across the cell membrane11 . BMDM cultures were all maintained at 37 °C in a 5% CO2 humidified incubator.
4
Bone Marrow-Derived Dendritic Cell Culture
5
Nos3 Signaling and Insulin Resistance
6
Dendritic Cell Stimulation by Microbial Agent
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For dendritic cell (DC) stimulation with MA, bone marrow (BM)-derived DCs (BMDCs) were prepared from BM suspensions from femurs and tibias of mice as described elsewhere [11 (link)]. Briefly, bone marrow cells were cultured with 20 ng/ml murine GM-CSF (R&D Systems) and 10 ng/ml murine IL-4 (R&D Systems) for 7 days. DCs were purified by positive selection using MACS Separator system (Miltenyi Biotec) with anti-CD11c monoclonal antibody (CD11c+ cells >90%). BMDCs were then cultured in the presence of plate-coated MA (0.01, 0.1, 1.0, and 10.0 μg/well) for 3 days and surface expression of CD80/86 and CD40 were examined by flow cytometry. Culture supernatants were examined for the production of cytokines and chemokine by ELISA.
7
Cultivation and Stimulation of Cell Lines
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293T cells were cultivated with complete DMEM medium (10% FCS, 2 mM L -glutamine, 10 U/ml penicillin-streptomycin). Ba/F3 and 32D cells were cultivated with complete RPMI 1640 medium (10% FCS, 2 mM L -glutamine, 10 U/ml penicillin-streptomycin) (all from Gibco, Thermo Fisher Scientific) supplemented with IL-3 (1 ng/ml; ImmunoTools). IL-3 stimulation was performed with 10 ng/ml IL-3 for 20 minutes.
The hSTAT5BN642H, hSTAT5B, and B6N WT T cells were isolated from LNs and spleens from 8- to 12-week-old mice. Following T cell activation by anti-CD3 (BD), T cells were grown in complete RPMI 1640 medium containing 10 mM HEPES, 1× MEM nonessential amino acids, 50 μM β-mercaptoethanol (all from Gibco, Thermo Fisher Scientific), 1 mM sodium pyruvate (MilliporeSigma), and 100 U/ml human IL-2 (ProleukinÒ; Novartis).
Cytokine stimulation of T cells was performed with human IL-2 (100 U/ml; ProleukinÒ; Novartis), murine IL-4 (100 ng/ml; R&D Systems), or murine IL-7 (10 ng/ml; R&D Systems).
The 293T and 32D cell lines were gifts of M. Hengstschläger (Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria) and F. Grebien (Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria), respectively. The Ba/F3 cell line was provided by A. D’Andrea (Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA).
The hSTAT5BN642H, hSTAT5B, and B6N WT T cells were isolated from LNs and spleens from 8- to 12-week-old mice. Following T cell activation by anti-CD3 (BD), T cells were grown in complete RPMI 1640 medium containing 10 mM HEPES, 1× MEM nonessential amino acids, 50 μM β-mercaptoethanol (all from Gibco, Thermo Fisher Scientific), 1 mM sodium pyruvate (MilliporeSigma), and 100 U/ml human IL-2 (ProleukinÒ; Novartis).
Cytokine stimulation of T cells was performed with human IL-2 (100 U/ml; ProleukinÒ; Novartis), murine IL-4 (100 ng/ml; R&D Systems), or murine IL-7 (10 ng/ml; R&D Systems).
The 293T and 32D cell lines were gifts of M. Hengstschläger (Center of Pathobiochemistry and Genetics, Institute of Medical Genetics, Medical University of Vienna, Vienna, Austria) and F. Grebien (Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria), respectively. The Ba/F3 cell line was provided by A. D’Andrea (Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA).
8
Lipid-based Nanoparticle Formulation and Characterization
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All lipids including 1,2-distearoyl-sn-glycero-3-phos-phocholine (DSPC; 20 mM), cholesterol (CHOL; 10 mM) and N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N- trimethyl ammonium methyl-sulfate (DOTAP; 20 mM) were purchased from Avanti Polar Lipid (Alabaster, USA). The fluorescent lipophilic dyes, 1,1_-dioctadecyl-3,3,3_,3_-tetramethylindocarbocyanine perchlorate (DiI) and 1,1_-dioctadecyl-3,3,3_,3_ tetramethylindodicarbocyanine (DiD) were purchased from Invitrogen (USA). Bovine serum albumin (BSA) was from Fluka (USA). 4-(2-hyd roxyethyl)-1 piperazineethanesulfonic acid (HEPES), Iscove’s Modified Dulbecco’s Medium (IMDM), Roswell Park Memorial Institute (RPMI) 1640 medium and phosphate-buffered saline (PBS; 155 mM NaCl, 1.5 mM potassium phosphate monobasic, 2.7 mM sodium phosphate dibasic, pH 7.2) were from Sigma (USA), human granulocyte macrophage colony-stimulating factor (GM-CSF), murine GM-CSF, human IL-4 and murine IL-4 were from R&D (USA). L-Glutamine was from Biosera. Anti-murine FITC conjugated CD11c and PE conjugated CD40, CD80, CD86, MHC-II and anti-human FITC conjugated CD14 and PE conjugated CD83, CD86 and HLA-DR antibodies were from BD Biosciences (USA). Lipopolysaccharide (LPS) was from Sigma (USA). Fetal calf serum (FCS) were purchased from Invitrogen (USA). CD14 human microbeads (Cat. No.#130-050-201) were purchased from Miltenyi Biotec (Germany).
9
Differentiating Bone Marrow DCs
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Bone marrow single-cell suspensions were cultured in complete medium (RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) supplemented with murine GM-CSF (10 ng/ml) and murine IL-4 (2 ng/ml) (R&D Systems, Minneapolis, MN). Fresh medium was added every 2 days. Nonadherent bone marrow DCs were harvested on day 6 for flow cytometry analysis.
10
Generation of Bone Marrow-Derived Dendritic Cells
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Bone marrow-derived DC were generated as previously described [28] (link). Briefly, bone marrow was harvested from femurs and tibias of wild type (WT), βc-/of BALB/c and C57BL/6 mice and passed through a nylon mesh to remove small pieces of bone and debris. Bone marrow cells were seeded at a density of 5 × 10 5 cells per 24-well in a differentiating culture medium consisting of IMDM (Gibco) with 10% FCS and 1% penicillin-streptomycin supplemented with 50 ng/mL murine GM-CSF (Renca-GM-CSF supernatant), 50 ng/mL murine IL-4 (R&D systems, Abingdon, UK) and 10 ng/mL murine Flt3-ligand (Renca-FLT3-L supernatant). On days 2 and 5, non-adherent cells were gently removed, and a differentiating culture medium was added. On day 7, the medium was replaced by a fresh medium containing 10 µg/mL LPS (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) or 10 µg/mL CpG (Enzo Life Sciences AG, Lausen, Switzerland) to induce DC maturation. Alternatively, DC cells were pulsed with OVA by suspending 10 6 cells/mL in a differentiating medium containing 10 µM OVA peptide SIINFEKL-K b . After a 4 h incubation period at 37 • C, with gentle shaking every 15 min, the OVA-pulsed DC were washed and resuspended in HBSS (Gibco) for injection in mice.
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