For TGF-β stimulation experiments LX-2 cells were seeded in 6-well plates at a density of 2–3 × 105 cells per well. Cells were serum starved overnight and transfected with miR-25 or control mimics, vs. miR-25 or control antagomirs, respectively, as described above. One day post transfection cells were treated with recombinant human TGF-β (5 ng/ml; Sigma) or an equal amount of phosphate-buffered saline (PBS) as control in serum-free medium. Cells were lysed after 6 h for subsequent protein analysis or after 24 h for RNA isolation.
Recombinant human tgf β
Manufactured by Merck Group
Sourced in Germany
Recombinant human TGF-β is a laboratory reagent produced using recombinant DNA technology. It is a member of the transforming growth factor beta protein family. The core function of this product is to serve as a signaling molecule in various cellular processes.
Automatically generated - may contain errors
3 protocols using recombinant human tgf β
1
TGF-β Stimulation of LX-2 Cells
2
Rat Hepatic Cell Line Response to TGF-β Stimulation
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The HSC-T6 immortalized rat cell line (Tumor Cell Bank of Chinese Academy of Medical Sciences, Beijing, China) was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin and 100 µg/ml streptomycin (Beijing Suolaibao Biotechnology Co., Ltd., Beijing, China). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. All experiments were carried out 24 h after the cells were seeded.
Cells were divided into two groups: The control group and the transforming growth factor (TGF)-β group. Recombinant human TGF-β (Sigma -Aldrich; Merck Millipore, Darmstadt, Germany) was used, since it is a major fibrogenic mediator in the liver (13 (link),14 (link)). TGF-β was administered to the cultured cells at 0, 1, 2, 5, 10 and 20 ng/ml for 0, 1, 3, 6, 12, 24 and 48 h. Cells were exposed to starvation conditions using FBS-free medium for 24 h prior to treatment with Recombinant human TGF-β.
Cells were divided into two groups: The control group and the transforming growth factor (TGF)-β group. Recombinant human TGF-β (Sigma -Aldrich; Merck Millipore, Darmstadt, Germany) was used, since it is a major fibrogenic mediator in the liver (13 (link),14 (link)). TGF-β was administered to the cultured cells at 0, 1, 2, 5, 10 and 20 ng/ml for 0, 1, 3, 6, 12, 24 and 48 h. Cells were exposed to starvation conditions using FBS-free medium for 24 h prior to treatment with Recombinant human TGF-β.
3
Marilyn Induced Treg Cells Protocol
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Bone marrow dendritic cells (BMDC) were generated as previously described.34 (link) Marilyn induced Treg (iTreg) cells were obtained from naive Rag2–/– Marilyn spleen cells (5 × 105 cells/ml) cultured with BMDC (105 cells/ml), HY-AbDby peptide (10 nm ), recombinant human TGF-β (2 ng/ml) and retinoic acid (1 μm , Sigma, St Louis, MO) for 5 days. Live cells were recovered on a gradient density (Lympholyte, Cedarlane Laboratories, Burlington, ON, Canada). Staining indicated ≥ 70% Foxp-3+ cells. Fresh BMDC (106 cells/ml) were co-cultured with iTreg cells (106 cells/ml) in the presence or absence of HY-AbDby peptide (1 nm ) for 48 hr.
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