Recombinant TGF-β, VEGF-A and the anti-VEGF-A-neutralizing antibody were from R&D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium (DMEM) and cell culture reagents were from Invitrogen (Carlsbad, CA, USA). All the other reagents were from Sigma (St. Louis, MO, USA) unless otherwise specified.
Recombinant tgf β
Manufactured by R&D Systems
Sourced in United States
Recombinant TGF-β is a laboratory product used for research purposes. It is a recombinant form of the transforming growth factor beta protein, which is involved in various cellular processes. This product is intended for use in experimental settings to study the biological functions of TGF-β.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant il 6, by R&D Systems (5 mentions)
Anti cd3, by BD (5 mentions)
Sb431542, by Merck Group (3 mentions)
Anti il 4, by R&D Systems (3 mentions)
Anti interferon γ, by BD (3 mentions)
Soluble anti cd28, by BD (3 mentions)
Macs separation column, by Miltenyi Biotec (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Tgf β, by R&D Systems (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Tgf β, by Merck Group (2 mentions)
Anti vegf a neutralizing antibody, by R&D Systems (1 mentions)
Vegf a, by R&D Systems (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Co2 incubator, by Thermo Fisher Scientific (1 mentions)
Tu177, by Thermo Fisher Scientific (1 mentions)
Amc hn 8, by Thermo Fisher Scientific (1 mentions)
Tu177, by R&D Systems (1 mentions)
Tu686, by Thermo Fisher Scientific (1 mentions)
37 protocols using recombinant tgf β
1
Quantifying Angiogenic Signaling Factors
2
Culturing and TGF-β Treatment of LSCC Cell Lines
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Three human LSCC cell lines (TU686, TU177 and AMC-HN-8) and 293T cells were purchased from BNBIO (Beijing, China) and preserved at the Otorhinolaryngology Head and Neck Surgery Biobank of Hebei Medical University. The TU686 and TU177 cells were cultured in RPMI-1640 medium (Gibco/Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco/Thermo Fisher Scientific, Inc.). The AMC-HN-8 and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco/Thermo Fisher Scientific, Inc.) supplemented with 10% FBS. The TU177 cells were treated with 10 ng/ml recombinant TGF-β (R&D Systems, Inc., Minneapolis, MN, USA) for 7 days and the medium was replenished every 2 days. All the cells were cultured at 37°C in a humidified 5% CO2 incubator (Thermo Fisher Scientific, Inc.).
3
Th17 Cytokine Profile Analysis
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To determine the levels of IFNγ, IL-17A, and IL-10 expressed under conditions of Th17 differentiation, murine splenocytes were cultured in RPMI 1640 medium supplemented with 5% (v/v) FBS. CD4-positive T cells were sorted employing CD4-coated magnetic beads and a magnetically activated cell sorting (MACS) separation column (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4-positive T cells were stimulated by addition of anti-CD3 (0.5 μg/mL) and soluble anti-CD28 Abs (1 μg/mL; both from BD Biosciences), anti-IFNγ (2 μg/mL) and anti-IL-4 (2 μg/mL) Abs (Invitrogen), recombinant TGF-β (2 ng/mL), and recombinant IL-6 (20 ng/mL) (R&D Systems, Minneapolis, MN, USA) for 3 days. Culture supernatants were subjected to sandwich ELISAs (R&D Systems). Alkaline phosphatase (Sigma) was used for color development. Absorbance was determined at a wavelength of 405 nm using an ELISA microplate reader (Molecular Devices, San Jose, CA, USA).
4
Examining Wnt Signaling Pathways
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The rat anti-WNT-5A (MAB645) antibody, recombinant WNT-3A (mouse; 1324, human; 5036), recombinant WNT-5A (mouse/human: 645), and recombinant TGF-β (human; 240) were obtained from R&D Systems. The mouse anti-BSA (SC57504) antibody was obtained from Santa Cruz Biotechnology, Inc. The horseradish peroxidase (HRP)–conjugated sheep anti–mouse antibody (NA931V), HRP-conjugated donkey anti–rabbit antibody (NA934V), and HRP-conjugated goat anti–rat antibody (NA935V) were purchased from GE Healthcare. Mouse anti–total β-catenin antibody was purchased from BD. Mouse anti-nonphosphorylated-β-catenin antibody (clone 8E7) was obtained from Merck. All other chemicals were of analytical grade.
5
Fibroblast Response to TGF-β Induction
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Mouse embryonic NIH/3T3 and human fetal lung MRC-5 fibroblasts were cultured at 37 °C in a 5% CO2 humidified environment and in DMEM supplemented with 10% fetal calf serum. Fibroblasts were then placed into 12-well culture plates at a density of 1 × 105 cells per well for later gene and protein expression assays. After incubating for 12 h in a serum-free media to induce serum starvation, fibroblasts were exposed to recombinant TGF-β (R&D Systems, Inc., Minneapolis, MN, USA) at a concentration of 10.0 ng/ml for 24 h (RNA collection) and 48 h (protein collection).
6
TGF-β Inhibitor Modulates NGF in IVD Cells
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To determine whether the TGF‐β inhibitor, SB431542, inhibits Ngf expression and NGF production in IVD cells, we examined the effect of SB431542 on TGF‐β-mediated Ngf expression and NGF production by IVD cells isolated from five mice. IVD cells were isolated using collagenase digestion, as described above. Disc cells were subsequently incubated in α‐minimal essential media (α‐MEM) with 10% fetal bovine serum in six‐well plates. One week later, IVD cells were stimulated with α‐MEM (vehicle), 10 ng/ml recombinant TGF‐β (Product no. 7666-MB, R&D Systems, Minneapolis, MN, USA), or 10 ng/ml recombinant TGF‐β + 1 µM SB431542 (Product no. S4317, Sigma Aldrich, St Louis, MO, USA) for 6 and 24 h. Thereafter, total mRNA was extracted and analyzed using qPCR. NGF concentration in the cell supernatant was determined using a commercial NGF ELISA kit (Product No. DY556, R&D Systems).
7
Coculture of CRC Cells and Stromal Cells
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CRC cells were cocultured with MSC, or normal skin fibroblasts as controls, at different ratios, for 5 days in tumor cell medium. In specific experiments, recombinant TGF-β (100 ng/mL, R&D Systems) or IL-6 (10 ng/mL, R&D Systems), the TGF-β inhibitors latency-associated peptide (LAP) (10 µg/mL, R&D Systems) or SB431542 (10 µg/mL, Sigma) or anti-IL-6 neutralizing antibodies (10 µg/mL, R&D Systems) were added to cultures as indicated. The lack of effect by the TGF-β inhibitors on basal E-cadherin expression was verified in preliminary experiments (data not shown). In experiments aimed at evaluating the role of cell-to-cell contact, MSC and tumor cells were plated in the upper and lower chambers, respectively, of transwell plates (0.4 µm pore size, Corning, Lowell, MA). Alternatively, tumor cells were cultured in the presence of MSC-conditioned medium harvested every 48 hr. Monocultures of MSC or tumor cells were used as controls. At the end of culture periods, supernatants were collected and cells were harvested and used for subsequent analyses.
8
Glucose Exposure and TGF-β Signaling in Kidney Cells
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HK-2 cells, human kidney proximal tubular epithelial cells, and MDCK cells, Madin Darby Canine Kidney cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Both cells were cultured in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5 ng/mL epidermal growth factor and 0.05 mg/mL bovine pituitary extract or Dulbecco’s modified Eagle’s medium (Mediatech Inc., Corning Subsidiary, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA), 100 μg/mL streptomycin, and 100 IU/mL penicillin at 37 °C in a 5% CO2 humidified air condition, respectively. Cells were incubated with 5.5 mM D-glucose (low-glucose, LG) or 55 mM D-glucose (high-glucose, HG) for the indicated period (24, 48, 72 h). To confirm the relationship between TGF-β and VSIG4, cells were stimulated using 10 ng/mL recombinant TGF-β (R&D systems, Minneapolis, MN, USA).
9
Fibroblast Culture and AAT Treatment Protocol
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Primary adult human lung fibroblasts were purchased from Lonza. Fibroblasts were cultured in Fibroblast Growth Medium supplemented with 0.5 mL recombinant human insulin, 0.5 mL hFGF-B, 0.5 mL GA-1000 (Gentamicin and Amphotericin B), 10 mL FBS and antibiotics (10,000 units/mL penicillin and 10,000 μg/mL streptomycin). Fibroblast cultures were maintained in collagen I (4 μg/mL)-coated wells in a humidified atmosphere with 5% CO2 at 37°C. Fibroblast cultures were tested in passages four to seven. Fibroblasts were cultured for 2 hours in serum-free media before supplementation with active plasma purified AAT (MyBioSource, Inc.) and 5 ng/ml recombinant TGFβ (R&D Systems). RNA was extracted from fibroblast for qRT-PCR. Primer sequences are provided in S1 Table .
10
Chondrogenic Differentiation Assay
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All reagents were from Sigma (Munich, Germany) unless otherwise indicated. Recombinant TGF-β was from R&D Systems (Wiesbaden, Germany). The anti-SOX9 (C-20) antibody was from Santa Cruz Biotechnology (Heidelberg, Germany), the anti-type-II collagen (II-II6B3) antibody from the NIH Hybridoma Bank (University of Iowa, Ames, USA), and the anti-type-X collagen (COL-10) antibody from Sigma. Biotinylated secondary antibodies and ABC were from Vector Laboratories (Grünberg, Germany). Luciferase activity was determined with the Luciferase Assay System (Promega GmbH, Mannheim, Germany) and normalized to total cellular proteins using the BCA protein assay kit (Pierce Thermo Scientific, Fisher Scientific GmbH, Schwerte, Germany). The Cell Proliferation Reagent WST-1 was from Roche Applied Science (Mannheim, Germany). The type-II collagen contents were measured with the native type-II collagen Arthrogen-CIA Capture ELISA kit (Chondrex, Redmond, WA, USA) and those for type-X collagen using a COL-10 ELISA (Antibodies-Online, Aachen, Germany).
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