CD4+ T cells were stimulated with anti-CD3 (2 μg/mL) (eBioscience), anti-CD28 (1 μg/mL) (eBioscience), IL-2 (50 ng/mL) (R&D Systems), and transforming growth factor β (20 ng/mL) (R&D Systems) and cultured for 3 days for Treg cell induction.
Transforming growth factor β
Manufactured by R&D Systems
Sourced in United States
Transforming growth factor β is a protein that regulates cellular processes such as cell growth, cell differentiation, and cell migration. It is a multifunctional cytokine that plays a role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd28, by Thermo Fisher Scientific (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Interleukin 2 (il 2), by R&D Systems (2 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (2 mentions)
Interleukin 6 (il 6), by BioLegend (2 mentions)
Interleukin 6 (il 6), by R&D Systems (2 mentions)
Il 10, by R&D Systems (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Af 401 na, by R&D Systems (1 mentions)
Mab406, by R&D Systems (1 mentions)
Macs magnetic column, by Miltenyi Biotec (1 mentions)
Timp 1, by R&D Systems (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Soluble rankl srankl, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Vitronectin coated plates, by STEMCELL (1 mentions)
13 protocols using transforming growth factor β
1
Treg Cell Induction Protocol
2
Th17 Differentiation from CD4+CD25- T Cells
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CD4+CD25− T cells were isolated from spleens of 6-month-old mice as described previously (25 (link)). Cells were purified using a MACS magnetic column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) with a CD4+ T cell enrichment kit (11-0041-82; eBioscience; Thermo Fisher Scientific, Inc.). The cells were then stimulated with a plate-bound anti-CD3e antibody (5 µg/ml; 11-0038-42, eBioscience; Thermo Fisher Scientific, Inc.) and anti-CD28 antibody (2 µg/ml; 16-0281-82, eBioscience; Thermo Fisher Scientific, Inc.). For Th17-cell polarization, the following exogenous cytokines and antibodies were added: Transforming growth factor-β (5 ng/ml, R&D Systems); anti-IL-4 antibody (46-7041-82; 10 µg/ml; eBioscience; Thermo Fisher Scientific, Inc.); IL-6 (MAB406; 30 ng/ml; R&D Systems); anti-IFN-γ antibody (MAB485; 10 µg/ml; eBioscience; Thermo Fisher Scientific, Inc.) and IL-1β (AF-401-NA; 20 ng/ml; R&D Systems). After 5 days of culture, the proportion of Th17 cells among all cells was determined by flow cytometry.
3
Plasma Protein Markers in RFCA
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Peripheral blood samples were taken before RFCA and the plasma levels of the following protein markers were measured using enzyme-linked immunosorbent assay kits: tissue inhibitor of metalloproteinases-1 (TIMP-1; R&D Systems, Minneapolis, MN, USA), transforming growth factor-β (R&D Systems, Minneapolis, MN, USA), and pro-atrial natriuretic peptide (pro-ANP; Biomedica, Antony, France).
4
Generating Langerin-expressing Epstein–Barr Virus-transformed BLCs
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Langerin expressing Epstein–Barr virus-transformed BLCs were used as autologous APC (van der Vlist et al., 2011 (link)) and were cultured in RPMI 1640 (InvitrogenTM) supplemented with 10% fetal calf serum (Biowittaker), 1% penicillin, and streptomycin (both Lonza). Langerin-expressing MUTZ-3-derived LCs were differentiated from the MUTZ-3 progenitor cell line with 100 ng/mL granulocyte-macrophage colony-stimulating factor (Biosource), 10 ng/mL transforming growth factor β (R&D Systems), and 2.5 ng/mL tumor necrosis factor α (Miltenyi) (de Jong et al., 2010 (link)).
5
Osteoclast Differentiation from PBMCs
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PBMCs isolated by density gradient centrifugation were plated in 96-well culture plates as described before.19 (link) PBMCs were left overnight for osteoclast precursors (OCPs) to adhere on bone slices (Immunodiagnostic Systems, Boldon, UK).20 (link) On the following day (day 1 of culture), medium was changed to Dulbecco's modified Eagle's Medium (DMEM) supplemented with macrophage colony stimulating factors (M-CSF) 25 ng/mL (Peprotech, London, UK). Three days later, medium was again changed to DMEM with M-CSF (25 ng/mL), soluble RANKL (sRANKL) (50 ng/mL; Peprotech), dexamethasone (10 nM; Sigma-Aldrich) and transforming growth factor β (2.5 ng/mL; R&D Systems) in order to differentiate the OCPs into mature OC.21 (link) The culture medium was then changed twice a week. Cells cultured on bone slices for 7, 14 and 21 days20 22 (link) were used for functional assays and gene expression.
6
Differentiation of hESCs and hiPSCs into Germ Layers
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H9 hESCs (WiCell, Madison, WI, USA) and the hiPSC lines FSPS13B, CF03, CF04, and CF05 were plated on vitronectin-coated plates (10 μg/mL, Stem Cell Technologies) and cultured in E6 media supplemented with 2 ng/mL transforming growth factor β (R&D) and 25 ng/mL fibroblast growth factor 2 (Dr. Marko Hyvönen, Cambridge University) making complete E8 media. Cells were maintained by weekly passaging using 0.5 mM EDTA (Thermo Fisher Scientific). The cells were differentiated into the three germ layers and functional cell types as previously described (Cheung et al., 2012 (link), Mendjan et al., 2014 (link)) and as described in the Supplemental Information .
7
Induction of Human Regulatory T Cells
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Human PBMCs were obtained from blood samples of healthy donors (Shanghai Blood Center) with ethical approval from Shanghai Blood Center Ethics Committee. Human CD4+CD25lowCD127highCD45RA+ naïve CD4+ T cells were sorted by BD FACS Aria II cell sorter and differentiated into inducible Treg cells in X-VIVO (Lonza, Malkersville, USA, Cat#04-418Q) medium supplemented with 10% fetal bovine serum (Invitrogen, Maltham, USA, Cat#10100147), 1% GlutaMAX, 1% sodium pyruvate, 1% minimum essential medium with nonessential amino acids, 1% penicillin-streptomycin, 100 U/mL IL2 (R&D systems, Minneapolis, USA, Cat#202-IL), and 5 ng/mL transforming growth factor-β (R&D Systems, Cat#240-B), in the presence of Dynabeads Human T-activator CD3/CD28 (Gibco, Cat#11 132D) at a bead-to-cell ratio of 1:4. Approximately 7 days later, the differentiation efficiency reached at least 90% and could be used for analysis.
8
Isolation and Th17 Differentiation of Murine CD4+ T Cells
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Naïve CD4+ T cells were isolated from the spleen of wild-type Balb/c mice via magnetic-activated cell sorting using a CD4+ T Cell Isolation Kit (Miltenyi Biotec, Germany), in accordance with the manufacturer’s instructions. The RPMI medium for stimulating Th17 cell differentiation included 10 μg/ml anti-CD3 (eBioscience, USA), 1.5 μg/ml anti-CD28 (eBioscience), 5 ng/ml transforming growth factor-β (R&D Systems, USA), and 20 ng/ml IL-6 (BioLegend, USA). After cells had been cultured in this conditioned medium for 2 days at 37°C, 100 μl of the supernatant was analyzed for mouse IL-17 (mIL-17) using an IL-17-specific enzyme-linked immunosorbent assay (ELISA). All tests were repeated three times independently, and the means and standard deviations (SDs) from three tests were calculated.
9
Isolation and Stimulation of Cardiac Fibroblasts
10
Th17 Cell Differentiation from Naive CD4+ T Cells
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Naïve CD4+ T cells were isolated from the spleen of WT Balb/c mice using magnetic activated cell sorting (MACS) CD4+ T Cell Isolation kit (Miltenyi Biotec, Cologne, Germany) according to the manufacturer’s instructions. The RPMI medium for stimulating Th17 cell differentiation included 1 μg/mL anti-CD3 (eBioscience, San Diego, CA, USA), 1 μg/mL anti-CD28 (eBioscience), 2 ng/mL transforming growth factor (TGF)-β (R&D systems, Minneapolis, MN, USA), 20 ng/mL IL-6 (BioLegend, San Diego, CA, USA), 5 μg/mL anti-IFN-γ (BioLegend) and 5 μg/mL anti-IL-4 (BioLegend). Cells were cultured in this conditioned medium for 3 days. At day 0, tIK protein or phosphate-buffered saline (PBS) as a control was added.
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