After removing the culture medium, resting macrophages were either incubated with fresh, serum-free RPMI to generate M0 or activated toward M1 or M2 phenotype by incubation for 48 h with either LPS (1 μg/ml, Sigma–Aldrich) and IFN-γ (10 ng/ml, Immunotools), or IL-4 (20 ng/ml, Immunotools) and IL-13 (5 ng/ml; Immunotools), respectively (Tedesco et al., 2015 (link)). To obtain CM, MDMs activated as described above were incubated for a further 72 h in serum-free RPMI without stimuli. Media were then collected and concentrated as described above for THP-1.
Il 13
Manufactured by Immunotools
Sourced in Germany
IL-13 is a recombinant human cytokine that functions as a regulator of inflammatory and immune responses. It is a protein that plays a role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Immunotools (8 mentions)
Lipopolysaccharides (lps), by Merck Group (5 mentions)
Inf γ, by Immunotools (3 mentions)
Ifn γ, by Immunotools (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Cell countess 3, by Thermo Fisher Scientific (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Fetal calf serum (fcs), by PAN Biotech (1 mentions)
Tgf 1, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Il 21, by Immunotools (1 mentions)
Anti human cd28 cd28.2, by BD (1 mentions)
Interleukin 7 (il 7), by Immunotools (1 mentions)
Il 33, by BioLegend (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Skim milk powder, by Merck Group (1 mentions)
Ficoll paque, by Merck Group (1 mentions)
Cli095, by Merck Group (1 mentions)
9 protocols using il 13
1
Macrophage Polarization and Conditioned Media
2
Jurkat and THP-1 Macrophage Activation
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Cell stimulation was done by incubating for 48 h 5 × 106 Jurkat or macrophage-derived THP-1 (resting M0) cells with basal medium, or medium samples collected from the basal side of VLC cultures at day 6–7 post-infection. Note that cell viability was assessed by trypan blue counterstaining using Cell countess 3 (Invitrogen) and was above 80% at the time of stimulation. Positive controls of cell activation were also included and generated as following. Jurkat cells activation was done by 48 h of phytohemagglutinin (Sigma, 10 μg/ml) and phorbol 12-myristate 13-acetate (Sigma–Aldrich, 20 ng/ml) stimulation. Differentiation of THP-1 into resting macrophage (M0) was done by incubation with phorbol 12-myristate 13-acetate (185 ng/ml, Sigma–Aldrich) for 24 h. Polarization of M0 into a macrophage with a pro-inflammatory phenotype (M1 positive control) was done by 48 h of INF-γ (20 ng/ml, Immunotools), LPS (100 ng/ml, Sigma–Aldrich), and phorbol 12-myristate 13-acetate (Sigma–Aldrich, 20 ng/ml) stimulation. Polarization of M0 into a macrophage with an anti-inflammatory phenotype (M2a positive control) was done by 48 h of IL-4 (20 ng/ml, Immunotools), IL-13 (20 ng/ml, Immunotools), and phorbol 12-myristate 13-acetate (Sigma–Aldrich, 20 ng/ml) stimulation.
3
Cytokine-Induced Differentiation of CD4+ T Cells
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Isolated CD4+ T cells were cultured in RPMI medium 1640 (Gibco) containing 10% FCS (Pan Biotech) and 1% penicillin/streptocmycin (Biochrom) for 3 days in the presence of recombinant interleukin (IL) 6 (20 ng/mL Immunotools), IL-7 (10 ng/mL, Immunotools), IL-9 (10 ng/mL, Immunotools), IL-13 (25 ng/mL, Immunotools), IL-21 (10 ng/mL, Immunotools), IL-33 (10 ng/mL, Biolegend), TGF-ß1 (20 ng/mL, R&D Systems), butyric acid (Roth), propionic acid (Roth), isobutyric acid (abcr), formic acid (Merck) or medium alone. Cells were stimulated with anti-human CD3 (OKT3, eBioscience) and anti-human CD28 (CD28.2, BD Pharmingen) at a final concentration of 1 μg/mL.
4
Jurkat and THP-1 Macrophage Activation
5
Macrophage Polarization Protocol
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Cell differentiation was induced via a 6-h exposure to 185 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma–Aldrich) in DMSO. Cells were then polarized toward M1 or M2 phenotype by incubation for 48 more hours with INF-γ (20 ng/ml, Immunotools) and LPS (100 ng/ml, Sigma–Aldrich) or with IL-4 (20 ng/ml, Immunotools) and IL-13 (20 ng/ml, Immunotools), respectively (Tjiu et al., 2009 (link); Maeß et al., 2014 (link)), still in the presence of PMA. Cells used for the resting condition were kept in the presence of PMA for 48 more hours in normal growth medium. CM were then prepared by keeping polarized as well as resting cells in serum-free RPMI without stimuli and no PMA for 72 more hours. Media were then collected and concentrated 10-fold using Amicon Ultra-15 centrifugal filter units with Ultracel-PL, cut-off 3 KDa (Millipore/Merck). Total protein content was then determined by Bradford assay, using bovine serum albumin as reference. CM were stored at -20°C until use.
6
Macrophage Polarization Assay
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RPMI 1640 was purchased from Lonza (Basel, Switzerland), and antibiotic solution (100 U/ml penicillin and 100 μg/ml streptomycin) was from Invitrogen Inc. (Carlsbad, CA, US). Monoclonal anti-human IκB-α, phospho-p38 mitogen-activated protein kinase (MAPK), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were from Cell Signalling (Danvers, MA, US). Horseradish peroxidase-conjugated secondary antibodies were from Vector (Peterborough, UK). Anti-CCR2 mAb, anti-CD163 mAb, anti-IL-1β mAb, brefeldin, Fix, and Perm buffer solutions were from eBioscience/Affymetrix (Santa Clara, CA, US); anti-CD80 mAb and anti-CD206 mAb were from BD Biosciences Pharmigen (San Diego, CA, US). The cOmplete™ inhibitor cocktail was from Roche Diagnostics (Mannheim, Germany). Fetal bovine serum (FBS), Ficoll-Paque (density 1.077 ± 0.001), Percoll, dexamethasone, curcumin, CLI-095, skim milk powder as well as other analytic grade chemical agents were from Sigma-Aldrich. Ultrapure LPS (LPS-EB), zymosan (ZYMO), Pam3CSK4, and Poly(I:C) were from InvivoGen (San Diego, CA, USA). IL-4, IL-13, and colony stimulating factor-1 (CSF-1) were from ImmunoTools (Friesoythe, Germany). The curcumin analogues GG6 and GG9 were synthesized as described elsewhere [13 (link)].
7
Murine Inflammation Induction Protocol
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LPS was purchased from Cayla Invivogen (Escherichia coli 0111:B4). Recombinant murine IFN-γ, IL-13 and IL-4 were purchased from Immunotools.
8
Differentiation of Monocytes to Myofibroblast-like Cells
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Blood samples were collected in EDTA tubes (BD Vacutainer) and processed within 24h. Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation on cell separation medium (Lympholyte, Cedarlane), followed by magnetic-activated cell sorting for CD14 using human microbeads and AutoMACS Pro device (Miltenyi Biotec). Cells were cultured in DMEM low glucose medium (Sigma) with the addition of 10% Foetal Bovine Serum (FBS) and 1% penicillin/streptomycin (both Gibco). To differentiate into myofibroblast-like cells, CD14+ monocytes were stimulated with 10 ng/ml TGFβ (PeproTech), 10 ng/ml IL-4, 10 ng/ml IL-10 and 10 ng/ml IL-13 (Immunotools) for 7 days.
9
Differentiation of Human Monocytes
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Human CD14+ primary Mo were isolated from freshly prepared buffy coat (Karolinska Institutet Biobank, Stockholm, Sweden) using a pluriBead cell separation kit (pluriSelect GmbH, Leipzig, Germany). Mo were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) in six-well culture plates at a density of 1 × 106/ml and exposed to NhhA, as indicated, to induce differentiation. Control cells were primed with M-CSF (50 ng/ml) for 5 days. A subset of these cells was then polarized for 24 h by either IFN-γ (10 ng/ml) or LPS (20 ng/ml; Sigma-Aldrich, St. Louis, MO) to M1Mφ or IL-4 (20 ng/ml) to M2Mφ. To generate DCs, Mo were cultured in GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) for 5 days followed by LPS (20 ng/ml) for 2 days to enhance maturation. M-CSF and GM-CSF were purchased from ProSpec-Tany TechnoGene Ltd. (Israel). IL-4, IFN-γ, and IL-13 were purchased from ImmunoTools (Germany).
In experiments including inhibitors, cytochalasin D (1 µM), PD98095 (10 µM), SP600125 (10 µM), Celastrol (500 nM), or SR11302 (1 µM) was added to the cell culture 30 min before NhhA stimulation. All inhibitors were purchased from InvivoGen (San Diego, CA) and tested for cytotoxicity using trypan blue and propidium iodide (PI) exclusion assays. Only inhibitor concentrations that ensured viability above 90% were used; control cells were treated with appropriate vehicles for the inhibitors.
In experiments including inhibitors, cytochalasin D (1 µM), PD98095 (10 µM), SP600125 (10 µM), Celastrol (500 nM), or SR11302 (1 µM) was added to the cell culture 30 min before NhhA stimulation. All inhibitors were purchased from InvivoGen (San Diego, CA) and tested for cytotoxicity using trypan blue and propidium iodide (PI) exclusion assays. Only inhibitor concentrations that ensured viability above 90% were used; control cells were treated with appropriate vehicles for the inhibitors.
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