Effects of extracellular succinate (50 µM and 500 µM) were studied on M1 and M2-like macrophages differentiated and polarized from the human monocytic cell line THP-1 as described before62 (link). THP-1 (ECACC, Sigma-Aldrich/Merck) monocytes were seeded at 5 × 105–1 × 106 cells per 6 well and stimulated for 72 h with 100 ng/ml Phorbol 12-myristate 13-acetate (PMA) in 1640-RPMI (Sigma-Aldrich, MI, USA) to induce adherence. They were put on cytokine and FCS-free 1640-RPMI media for 24 h before differentiated into M1 macrophages by 100 ng/ml LPS (Sigma-Aldrich, MI, USA) and 20 ng/ml IFNɣ (Biolegend, CA, USA) and into M2 macrophages by using 20 ng/ml IL-4 (Miltenyi, Germany) and 10 ng/ml IL-13 (Miltenyi, Germany) for 72 h. Polarization state and transcriptional changes upon succinate stimulation were confirmed by qPCR. Primary human CD14 + cells were freshly isolated from human blood (commercially acquired buffy coats) by density-gradient isolation and MACs (Miltenyi, Germany) and seeded at 1 × 106 cells per well before stimulated with LPS and succinate (Sigma-Aldrich, MI, USA). IL-1β secretion was analyzed in the culture media after 24 h by human-Interleukin 1β Quantikine ELISA (R&D Systems, MN, USA).
Il 13
Manufactured by Miltenyi Biotec
Sourced in Germany, France
IL-13 is a laboratory product offered by Miltenyi Biotec. It is a recombinant human interleukin-13 protein. Interleukin-13 is a cytokine that plays a role in immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Miltenyi Biotec (4 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Lipopolysaccharides (lps), by Miltenyi Biotec (2 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Ecacc, by Merck Group (1 mentions)
Bradford based assay, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Trypsin, by Harvard Bioscience (1 mentions)
Collagenase, by Merck Group (1 mentions)
Human il 4, by Miltenyi Biotec (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Harvard Bioscience (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Dispase 2, by Roche (1 mentions)
Eht1864, by Bio-Techne (1 mentions)
Basic fibroblast growth factor (bfgf), by Miltenyi Biotec (1 mentions)
Pdgf bb, by Miltenyi Biotec (1 mentions)
Edu staining proliferation kit ifluor 488, by Abcam (1 mentions)
Il 17, by Miltenyi Biotec (1 mentions)
9 protocols using il 13
1
Succinate Modulates Macrophage Polarization
2
Cytokine Stimulation of Immune Cells
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HLMs, monocytes, and MDMs were cultured in 24-well plates in RPMI 1640 supplemented with 5% FBS (Sigma-Aldrich, Milan, Italy), 2 mM l-glutamine, and 1% antibiotic-antimycotic solution.
The cells were treated with IL-13 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), IL-4 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), detoxified LPS (100 ng/mL) (from Escherichia coli serotype 0111:B4; Sigma-Aldrich, Milan, Italy), or TSLP (5 ng/mL) for 16 h or 6 h at 37 °C. In selected experiments, the cells were preincubated (30 min, 37 °C) with or without actinomycin D (1 μg/mL) and then stimulated (16 h, 37 °C) with LPS or IL-4. At the end of incubation, the supernatants were collected and stored at −80 °C for subsequent ELISA quantification of cytokines. Lysis of the cells in the plates was carried out by using 0.1% Triton X-100 for total protein quantification by a Bradford-based assay (Bio-Rad, Segrate, MI, Italy).
The cells were treated with IL-13 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), IL-4 (10 ng/mL) (Miltenyi Biotec, Bologna, Italy), detoxified LPS (100 ng/mL) (from Escherichia coli serotype 0111:B4; Sigma-Aldrich, Milan, Italy), or TSLP (5 ng/mL) for 16 h or 6 h at 37 °C. In selected experiments, the cells were preincubated (30 min, 37 °C) with or without actinomycin D (1 μg/mL) and then stimulated (16 h, 37 °C) with LPS or IL-4. At the end of incubation, the supernatants were collected and stored at −80 °C for subsequent ELISA quantification of cytokines. Lysis of the cells in the plates was carried out by using 0.1% Triton X-100 for total protein quantification by a Bradford-based assay (Bio-Rad, Segrate, MI, Italy).
3
Macrophage Polarization Protocol
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After 7 days of differentiation, cells were washed twice with sterile PBS. For M1-activation, 2 mL RPMI 1640/5% FCS containing 1 μg/mL LPS (Sigma-Aldrich Chemie GmbH, Munich, Germany) + 300 U/mL IFNy (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to the wells. M2-activation was induced by the addition of medium containing 40 ng/mL IL-13 (Miltenyi Biotec, Bergisch Gladbach, Germany) + 40 ng/mL IL-4 (Miltenyi Biotec, Bergisch Gladbach, Germany) instead of LPS + IFNy. Control cells were cultured with RPMI 1640/5% FCS medium only. Cells were then incubated for 24 h under these conditions as described by Mosser and Zhang [26 (link)]. In order to demonstrate successful macrophage polarization, culture samples were stained with anti-CD206 (to assess M2-polarization) and anti-iNOS (to assess M1 polarization). Representative histograms are shown in Supplementary Figure 1 in Supplementary Material available online at https://doi.org/10.1155/2017/7584621 .
4
Isolation and Stimulation of Dermal Cells
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Human skin biopsies were incubated with 2.2 U/mL dispase II (Roche) overnight at 4 °C. After removal of the epidermal layer, the dermis was digested with 2.5 mg/mL collagenase (Sigma, Darmstadt, Germany) for 45 min at 37 °C. Cell suspension was passed through a 70 µm filter. Cells were cultured at 37 °C, 5% CO2 in DMEM medium (Anprotec, Bruckberg, Germany) containing 10% fetal calf serum (FCS; PAN-Biotech, Aidenbach, Germany) and 1% penicillin/streptomycin (Biochrom, Berlin, Germany). After reaching confluence, cells were passaged using 0.05% trypsin and 0.02% EDTA (Biochrom). Cells were stimulated with 10 ng/mL human IL-4 (Miltenyi, Bergisch-Gladbach, Germany), IL-6 (Miltenyi), IL-13 (Miltenyi), or TNFα/IL1β (Miltenyi) or 20 ng/mL TGFβ1 (Peprotech, Hamburg, Germany) in DMEM/0.5 % FCS for 9d. Medium was changed after 7d. To exclude any effects of proliferation or cell death, the cell number was counted, and the sThy-1 concentration was presented as pg per 10,000 cells.
5
Proliferative signaling in human airway smooth muscle cells
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Primary aSMCs were isolated from human bronchial biopsies. Additional detail on the method is provided in an online data supplement . Human aSMCs were seeded into 24-well plates (10 000 aSMCs/well) and allowed to adhere during 6 hour before serum starvation during 24 hours. When indicated, human aSMCs were treated with the Rac inhibitor, EHT1864 (10−5 M; Tocris Bioscience), P21-activated kinases (Pak) inhibitor IPA3 (10−5 M; Tocris Bioscience), Akt inhibitor VIII (10−5 M; Calbiochem), MEK inhibitor PD98059 (10−5 M; ThermoFisher), JAK inhibitor ruxolitinib (10−5 M; InvivoGen) added 30 min before stimulation with bFGF (25 ng/mL; Miltenyi Biotech), PDGF-bb (25 ng/mL; Miltenyi Biotech), interleukins (IL)-13 (10 ng/mL; Miltenyi Biotech), IL-33 (10 ng/mL; Miltenyi Biotech), IL-17 (20 ng/mL; Miltenyi Biotech), IL-9 (10 ng/mL; Miltenyi Biotech) or TSLP (10 ng/mL; Miltenyi Biotech) for 48 hours. Cells were stained with EdU for 12 hours at 10−5 M according to the manufacturer’s indications (EdU Staining Proliferation Kit iFluor 488, ID: ab219801, Abcam). Proliferation was quantified by the ratio of EdU-positive cells over the total number of cells. Proliferative signalling pathways were analysed by immunoblotting detailed in an online data supplement .
6
Macrophage Differentiation and Polarization
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For BMDC differentiation into BM-derived macrophages (BM-Mφs), BMDCs were cultured for an additional 7 days in the same medium supplement with 10 ng/ml recombinant macrophage colony-stimulating factor (M-CSF) (Peprotech). The efficiency of differentiation was confirmed by FACS analysis for CD11b and F4/80 antigen surface expression (Supplementary Fig. S5 ). To induce the macrophage phenotypic polarization, WEHI274.1 cells and BM-Mφs were treated for 24 hours with 100 ng/ml IFN-γ (Peprotech) or 200 ng/ml LPS (SIGMA) for M1Mφs and 20 ng/ml IL-4 (R&D systems) or 20 ng/ml IL-13 (Miltenyi Biotec) for M2Mφs.
7
Hospicell Immunomodulatory Cytokine Regulation
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Hospicells were incubated with complete RPMI 1640 (PAA) supplemented with 5% fetal calf serum (PAA). For induction of HLA-G, IL-1β (10 ng/ml), IL-6 (10 ng/ml), TGF-β (10 ng/ml), IGF (200 ng/ml), VEGF (50 ng/ml), FGF (10 ng/ml), IL-13 (100 ng/ml), IL-2 (100 ng/ml), and EGF (10 ng/ml) were used (all from Miltenyi Biotec, France). For inhibition of HLA-G, Janus kinase inhibitor (JAK inhibitor I; Calbiochem, France), STAT3 inhibitor (Calbiochem, France), NF-κB inhibitor (Merck-Chemicals, France), and mitogen-activated protein kinases (MAP-kinases; MPK) inhibitor were applied (Sigma, France) at 1 μM.
8
Isolation and Differentiation of Human Macrophages
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Human monocytes were obtained from 6 healthy volunteers. Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood by density gradient centrifugation on Lympholyte Cell Separation Media (Cedarlane Laboratories Limited, Burlington, ON, Canada). CD14+ monocytes were separated from PBMCs by immunomagnetic sorting using anti-CD14 (MACS CD14 Microbeads; Miltenyi Biotec, Auburn, CA, USA) magnetic microbeads [21 (link)]. Immunomagneting sorting efficiency was 98% according to flow cytometry analysis (data not shown). CD14+ monocytes were immediately subjected to macrophage differentiation, as described by Tarique and co-workers [22 (link)]. Briefly, CD14+ monocytes were cultured in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% FBS, 1% penicillin/streptomycin (Sigma-Aldrich, USA), 10 mM HEPES (Euroclone, Pero MI, Italy) and 1 mM L-glutamine (Lonza, USA). M1-like Macrophage differentiation was performed with 50 μg/mL GM-CSF (Miltenyi Biotec, Auburn, CA, USA) for 5 days, followed by 4 days of 20 μg/mL LPS + 20 μg/mL IFNγ (Miltenyi Biotec, Auburn, CA, USA) treatment. M0-like macrophages were obtained after 50 μg/mL GM-CSF treatment for 9 days, while M2-like macrophages were differentiated with 50 μg/mL GM-CSF (Miltenyi Biotec, Auburn, CA, USA) for 5 days, followed by 20 μg/mL IL-4 + 20 μg/mL IL-13 (Miltenyi Biotec, Auburn, CA, USA) treatment for 4 days.
9
Monocyte-Derived Dendritic Cell Isolation and Activation
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Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (blood donors from EFS, Toulouse) by Ficoll separation (PAA). Monocytes were isolated from PBMC with the use of CD14 microbeads and a magnetic bead separation system (Miltenyi, Paris, France). Monocytes were maintained for 5 days in RPMI medium supplemented with 10% FCS, 1% penicillin-streptomycin, 20 ng/mL recombinant GM-CSF (Miltenyi, Paris, France) and 50 ng/mL IL-13 (donated by Sanofi-Aventis). On day 5, non-adherent moDC were recovered and stimulated for 24 h with either S. aureus or S. epidermidis secretome at 5% (v/v), or LPS at 100 ng/mL or ADE (lg/mL) as indicated. Cells were stained and submitted to flow cytometry analysis on a FACSCalibur cytometer (BD Biosciences, Le Pont de Claix, France) as detailed in the flow cytometry analysis section.
cells were isolated from PBMC with the use of naive CD4 þ T cell isolation kit II and CD4 T cell isolation kit II (Miltenyi, Paris, France). For proliferation assays, CD4 þ T cells were stained with CFSE (Invitrogen, Waltham, MA) and cocultured with allogeneic moDC as indicated for 5 days in 96-well plates at a ratio of 1 stimulator to 10 T cells. Proliferation (%) was assessed by flow cytometry.
cells were isolated from PBMC with the use of naive CD4 þ T cell isolation kit II and CD4 T cell isolation kit II (Miltenyi, Paris, France). For proliferation assays, CD4 þ T cells were stained with CFSE (Invitrogen, Waltham, MA) and cocultured with allogeneic moDC as indicated for 5 days in 96-well plates at a ratio of 1 stimulator to 10 T cells. Proliferation (%) was assessed by flow cytometry.
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