Some cells were treated with 5 ng/mL of recombinant human TGFβ (PeproTech) and 5 ng/mL of recombinant human TNFα (PeproTech) as metastatic inducers for the indicated time periods. When cells with genetic suppression of a gene of interest were also treated with TGFβ1 and TNFα, the cells were transduced and selected as described below and then treated with 5 ng/mL of recombinant human TGFβ (PeproTech) and 5 ng/mL of recombinant human TNFα (PeproTech).
Recombinant human tgf β
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Recombinant human TGF-β is a protein that functions as a growth factor. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
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Anti cd28, by BioLegend (4 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (2 mentions)
Anti cd3ε, by BioLegend (2 mentions)
Anti cd3, by BD (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
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Recombinant rat il 6, by Thermo Fisher Scientific (2 mentions)
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Anti ifn γ antibody, by Thermo Fisher Scientific (2 mentions)
Anti il 4 antibody, by BioLegend (2 mentions)
Anti ifn γ, by BioLegend (2 mentions)
Recombinant human tnf α, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
57 protocols using recombinant human tgf β
1
Metastatic Induction by TGFβ and TNFα
2
Preparation of Stock and Working Solutions
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A stock solution of 100 mM for each compound was prepared by dissolving in DMSO and stored at −20 °C until use. Working solution (treatment) was prepared before use by diluting the stock solution in the culture media. Thus, the final concentration of DMSO in the medium did not exceed 0.04% for 40 μM, and 0.02% for 20 μM of treatment. Human recombinant TGFβ was purchased from Peprotech (NJ 08553, USA), reconstituted according to the manufacturer's instruction, and stored at −20 °C until use. Anti-collagen I (ab34710), anti-alpha smooth muscle actin (ab5694), AlexaFluor 488 labeled donkey anti-rabbit (ab150073) antibodies were purchased from Abcam (Tokyo, Japan). ProLong Diamond antifade mountant with 4′,6-Diamidino-2-Phenylindole (DAPI: double stranded DNA staining) was purchased from Invitrogen (ThermoFisher, USA).
3
Fibrosis Markers in Tubular Epithelial Cells
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Primary human tubular epithelial cells (hTECs) were isolated from the noncancerous part of the human kidney tissue obtained from kidneys that were surgically removed due to renal cell carcinoma. hTECs were cultured for four to six passages in EGM-2 MV medium (Clonetics Co., San Diego, CA, USA) supplemented with 10% fetal bovine serum, 1 mg/mL hydrocortisone, 12 mg/mL bovine brain extract, 50 mg/mL gentamycin, 50 ng/mL amphotericin B, and 10 ng/mL epidermal growth factor. The cells were then stimulated with human recombinant TGF-β (2 ng/ml; PeproTech, Rocky Hill, NJ, USA) in the presence or absence of different concentrations (2–4 μΜ) of SB-731445. Microscopic morphological changes were observed between the groups. Quantitative real-time PCR for fibrosis markers, including collagen-1, fibronectin, and periostin, was carried out using an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). All in vitro experiments were repeated 3 times to confirm the reproducibility.
4
TGF-β-mediated Activation of hPSCs
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To examine the expression of FGF receptors and differentiation/activation markers in TGF-β-mediated hPSC activation, cells were seeded into a 12 well plate at a seeding density of 50,000 cells/well. After 24h, cells were starved for 24 h, then treated with human recombinant TGF-β (Peprotech, Hamburg, Germany). After 24 h of the incubation, cells were lysed and total RNA were isolated using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma Aldrich) and the RNA concentration was measured using a NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific, Waltham, USA). cDNA was synthesized with iScript™ cDNA Synthesis Kit (BioRad, Veenendaal, The Netherlands), and 10 ng cDNA were used for each PCR reaction. The real-time PCR primers (Table 1 ) were purchased from Sigma Aldrich. Quantitative real time PCR was performed with 2x SensiMix SYBR and Fluoroscein Kit (Bioline GmbH, Luckenwalde, Germany) using a BioRad CFX384 Real-Time PCR detection systems (BioRad). Gene expression levels were normalized to the expression of the house-keeping gene 18s rRNA.
5
Immunofluorescent Characterization of Pericytes
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Primary human brain vascular pericytes were obtained from ScienCell (Carlsbad, CA) and grown according to the vendor's instructions. Human recombinant TGF-β was purchased from Peprotech (100-21). Antibodies used in the immunofluorescent staining were purchased from commercial sources as follows: rabbit-anti-mouse collagen IV (Millipore, AB756P), mouse-anti-mouse-α-SMA-FITC (Sigma, F3777), goat-anti-mouse CD31 (R&D, AF3628), Alexa Fluor 555 conjugated goat-anti-Rabbit Ig (Invitrogen, A21429) and Alexa Fluor 488 conjugated goat-anti-human Ig (Invitrogen, A11013). Two monoclonal anti-CD248 antibodies, Clone 8 and 9G5, were generated in house by immunization of rabbits or rats (respectively) with a CD248ECD-Fc fusion protein. These antibodies were selected for all immunostaining and internalization assays as they do not compete with MORAb-004 for CD248 binding in a competition FACS assay (Figure S1C ).
6
T Cell Culture Protocol with Cytokines
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T cell medium consisted of RPMI 1640 medium, supplemented with 10% heat-inactivated FCS, penicillin (100 IU/mL) and streptomycin (100 IU/mL) (Invitrogen, Ghent, Belgium). Purified anti-CD3 (UCHT1), anti-CD28 (37407) and human recombinant IL-1β were from R&D Systems (Minneapolis, USA). Human recombinant IL-2, human recombinant IL-6, and human recombinant TGF-β were from PeproTech (Rocky Hill, USA). TX527 [19-nor-14,20-bisepi-23-yne-1,25(OH)2D3], a 1,25(OH)2D3-analog, was synthesized by M. Vandewalle and P. De Clercq (University of Ghent, Ghent, Belgium) and obtained from Théramex S.A. (Monaco, France). 1,25(OH)2D3 (calcitriol) was from Sigma-Aldrich (St. Louis, USA).
7
Exosome-Mediated Regulation of Cardiac Fibroblast Transcription
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To measure the activity of the TCF/LEF and AP-1 responsive elements in cardiac fibroblasts, TCF/LEF-hCF and AP1-hCF reporter cardiac fibroblasts were stimulated with 5 μL of the exosome stock diluted in 100 μL culture medium, 100 μL conditioned medium or 10 ng/mL human recombinant TGF-β (Peprotech, Rocky Hill, USA) for 24h. Luciferase expression in the reporter cells was assessed using the Bright-Glo Luciferase Assay System (Promega, Madison, USA). Bioluminescence was measured by SyneryHT plate reader (BioTek, Winooski, USA) or Wallac Victor2 1420 Multilabel counter. (Perkin-Elmer, Waltham, USA).
8
Chrysotile Exposure and TGF-β Effects
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Dose- and time-dependence experiments were performed to determine the appropriate concentration and time, respectively, for incubating MeT-5A cells with chrysotile. As a consequence of these preliminary results, 4.5 × 105 cells in 100-mm-diameter Petri dishes were incubated for 72 h in the absence or presence of 5 µg/cm2 of chrysotile asbestos suspension or 10 ng/μL TGF-β solution in M199 medium. The protein content in the cells was detected using a bicinchoninic acid assay (BCA) kit (Sigma Chemical Co., Saint Louis, MO, USA). The plasticware for cell culture was provided by Falcon (Corning Incorporated, Corning, NY, USA). Ultrapure water (Millipore, Burlington, MA, USA) was used for all experiments. A solution of recombinant TGF-β was obtained by re-suspending the lyophilized form of Human Recombinant TGF-β (PeproTech, Rocky Hill, CT, USA) in a 10 mM citric acid solution, with a pH of 3.0 in order to increase the storage time. The solution was then diluted in a 0.1% BSA-based buffer.
9
Collagen Gel Contraction Assay
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A collagen suspension (5 ml) containing 3.0 ml of collagen G1 (5 mg ml−1, Matrix Biosciences, Morlenbach, Germany), 0.5 ml of 10 × M199 medium (Sigma), 85 μl of 1 N NaOH (Sigma) and sterile water was mixed with 1.0 ml (2 × 106) of cells. The collagen gel and cell suspension (0.6 ml per well) was plated in a 24‐well culture plate and was allowed to polymerize for 1 h at 37 °C. For the effect studies, polymerized gel was incubated with 1 ml of 0.5% FBS-containing medium with or without human recombinant TGFβ (5 ng ml−1) (Peprotech, Rocky Hill, NJ, USA) together with 10 μM LDE225 (Erismodegib, Selleckchem, Boston, NY, USA) followed by detachment of the gels from the culture wells. For other experiments, medium with or without TGFβ was added before gel detachment. Photographs were made with a digital camera at different time points (0, 24, 48 and 72 h). The size of the gels was digitally measured and normalized with their respective well size in each image. Gel contraction experiments were performed in duplicate in three independent experiments.
10
Modulating Notch and TGF-β in Lung Cancer Cell Lines
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Human bronchial epithelial Beas2b cells were a kind gift of Dr. John Langenfeld, A549, H1299, H358, H1650, H322, H441, H1975, H838, H1838, H23, and H1755 cell lines were obtained from the American Type Culture Collection (ATCC), and Sk-Lu-1, H2030, and HCC-44 cell lines were a gift from Bristol Myers Squibb (Princeton, NJ). Cells were cultured in media as recommended by ATCC and maintained at 37° with 5% CO2 and ambient O2. For EDTA-induced Notch activation, cells were exposed to 1 mM EDTA or culture media for 30 min, then placed back in culture media for 1 hour. For inhibition of Notch activation, cells were treated with 1 μM RO4929097 or DMSO for 48 hours. Human recombinant TGF-β (PeproTech) was used at a concentration of 10 ng/ml for 48 hours. TGF-β inhibitor S431542 (Sigma) was used at a concentration of 10 μM for 48 hours.
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