Human-derived fibroblasts from healthy and asthma subjects were maintained in DMEM medium supplemented with 2 μg/mL of insulin, 1 mM of sodium pyruvate, 1 mM of nonessential amino acids, 4 mM of glutamine, 10% fetal calf serum, and antibiotics (penicillin/streptomycin) at 37°C and 5% CO2. Cells were seeded at 0.5 − 1 × 105 cells/mL in 25 cm2 flasks. At ~70% confluency, cells were stimulated with 20 ng/ml of IL-13 (ProSpec). Control cell cultures were either left untreated or treated with equal volumes of DMSO as vehicle.
Il 13
Manufactured by ProSpec
Sourced in Israel
IL-13 is a lab equipment product that measures the concentration of Interleukin-13 (IL-13) in biological samples. IL-13 is a cytokine involved in various immune and inflammatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by ProSpec (4 mentions)
Ifn γ, by ProSpec (3 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Gm csf, by ProSpec (2 mentions)
M csf, by ProSpec (2 mentions)
Interleukin 6 (il 6), by ProSpec (2 mentions)
Il 10, by ProSpec (2 mentions)
Tgf β1, by R&D Systems (1 mentions)
Tnf α, by ProSpec (1 mentions)
Il 1β, by ProSpec (1 mentions)
Transforming growth factor β1, by R&D Systems (1 mentions)
Interferon ifn γ, by ProSpec (1 mentions)
Ds fi2 camera, by Nikon (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
M csf, by R&D Systems (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Mirna, by GenePharma (1 mentions)
Penicillin g, by Thermo Fisher Scientific (1 mentions)
6 protocols using il 13
1
Cultivation and Stimulation of Human Fibroblasts
2
Dendritic Cell Cytokine Culture
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Recombinant human cytokines used for DC treatments were as follows: LT-α (TNF-β), GM-CSF, M-CSF, IL-1β, IL-6, IL-4, TNF-α, IL-13, IFN-γ, IL-10 (ProSpec-Tany Technogene, Rehovot, Israel) and TGF-β1 (R&D Systems, Minneapolis, Minnesota, USA) were used in culture within a final concentration range of 5–80 ng/ml. Dexamethasone was used at a final concentration in culture at 30 ng/ml (Sigma-Aldrich, St. Louis, MO). All cell culture experiments utilized RPMI 1640 tissue culture medium, heat-inactivated (56°C/20 min) fetal calf serum (FCS), penicillin/streptomycin and L-glutamine (SAFC Biosciences, Lenexa, KS).
3
Differentiation of Macrophage Subtypes
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Recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage (M)-CSF, interleukin (IL)-1β, IL-6, IL-4, IL-10, IL-13, interferon (IFN)-γ, lymphotoxin (LT)-α, tumor necrosis factor (TNF), M-CSF and GM-CSF were purchased from ProSpec-Tany Technogene (Rehovot, Israel), transforming growth factor (TGF)-β1 from R&D Systems (Minneapolis, MN), and dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO). A mouse monoclonal antibody (clone 3C9) that recognizes the IgV domain of human CRIg was kindly provided by Dr. Menno van Lookeren Campagne (Genentech, San Francisco, CA). RPMI 1640 tissue culture medium, foetal calf serum (FCS) and L-glutamine were purchased from SAFC Biosciences (Lenexa, KS).
4
Macrophage Polarization by Cancer Cell EVs
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Human primary CD14+ monocytes were isolated from a buffy coat and tested for purity using flow cytometry. Monocytes were seeded at a density of 20 × 104 in a 24-well plate. The culture medium was advance RPMI-1640 (Gibco) supplied with 10% FBS, 1% L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml fungizone. The cells were allowed to adhere for 90 min, and unattached cells were then washed away with PBS and the wells were supplied with fresh media plus 100 ng/ml M-CSF (R&D Systems, Minneapolis, MN, USA). Monocytes were allowed to differentiate into macrophages for seven days, and images were taken each day using a Nikon DS-Fi2 camera (Nikon, Tokyo, Japan).
To study the effect of cancer cell EVs on macrophage polarization, differentiated macrophages were then subjected to EVs isolated from HSC-3 and SCC-25 cells for 24 hours. As a positive control, macrophages were differentiated to M1 macrophages using 10 ng/ml LPS (Sigma-Aldrich) and 20 ng/ml IFN-γ (Prospec, Rehovot, Israel), and to M2 macrophages using 20 ng/ml IL-4 and 20 ng/ml IL-13 (Prospec).
To check macrophage phenotypes using q-PCR, cells were lysed using an RLT buffer (Qiagen, Düsseldorf, Germany) and kept at −80°C until RNA isolation.
To study the effect of cancer cell EVs on macrophage polarization, differentiated macrophages were then subjected to EVs isolated from HSC-3 and SCC-25 cells for 24 hours. As a positive control, macrophages were differentiated to M1 macrophages using 10 ng/ml LPS (Sigma-Aldrich) and 20 ng/ml IFN-γ (Prospec, Rehovot, Israel), and to M2 macrophages using 20 ng/ml IL-4 and 20 ng/ml IL-13 (Prospec).
To check macrophage phenotypes using q-PCR, cells were lysed using an RLT buffer (Qiagen, Düsseldorf, Germany) and kept at −80°C until RNA isolation.
5
Isolation and Culture of Murine Macrophages
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BMDMs were isolated from 6 to 8-week-old BABL/c male mice as described (Davis, 2013 (link)). Primary cells were maintained on bacterial grade plates for 1 week in DMEM (Hyclone), supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine, 100 units/mL penicillin G, 100 mg/mL streptomycin (Invitrogen) and 5 ng/mL M-CSF (ProSpec). Adherent cells were then replated on plastic tissue culture plates in fresh media and used 24 h after replating. The immortalized rat Kupffer cell line (purchased from Applied Biological Materials Inc.) was maintained in the same media as BMDMs but without M-CSF.
For stimulation, BMDMs or the Kupffer cell line were exposed to recombinant IFN-γ, IL-10, IL-4 or IL-13 (ProSpec) after starvation overnight in 0.5% fetal bovine serum (Hyclone), and harvested at the appropriate time points.
For transfection, 40 nM miRNA mimics, inhibitors or negative control (GenePharma, China) was transfected using the Lipofectamine 2000 Transfection Reagent (Life technologies) according to the manufacturer's instructions.
For stimulation, BMDMs or the Kupffer cell line were exposed to recombinant IFN-γ, IL-10, IL-4 or IL-13 (ProSpec) after starvation overnight in 0.5% fetal bovine serum (Hyclone), and harvested at the appropriate time points.
For transfection, 40 nM miRNA mimics, inhibitors or negative control (GenePharma, China) was transfected using the Lipofectamine 2000 Transfection Reagent (Life technologies) according to the manufacturer's instructions.
6
Macrophage Differentiation and Polarization
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The human THP-1 and HEK293T cell lines were obtained from the iCell Bioscience Inc. (Shanghai, China). THP-1 cells were cultured in RPMI-1640 medium (Gibco, MD, USA) supplemented with 15% fetal bovine serum (FBS, Gibco, MD, USA), 1% penicillin/streptomycin solution (Gibco, MD, USA) and 50 μM β-Mercaptoethanol (Solarbio, Beijing, China) in a humidified cell incubator with 5% CO2 at 37°C. 293T cells were grown in Dulbecco’s modified Eagle’s medium with high glucose (DMEM, Gibco, MD, USA) with 10% FBS and 1% penicillin/streptomycin. For M0 macrophage differentiation, THP-1 cells were treated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Solarbio, Beijing, China) for 24 h. For M1 polarization, THP1-derived M0 macrophages were then treated with 20 ng/mL IFNγ (Prospec, NZ, Israel) and 100 ng/mL lipopolysaccharides (LPS, Solarbio, Beijing, China) for another 48 h. For M2 polarization, THP1-derived M0 macrophages were treated with 20 ng/mL IL4 and 5 ng/mL IL13 (Prospec, NZ, Israel) for 48 h.
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