Mouse splenic CD4+ T lymphocytes were isolated using a mouse CD4+ T Cell Isolation Kit (Miltenyi Biotec; Auburn, CA, USA) with magnetic beads following the manufacturer’s instructions. Mandibular BM-MSCs at passage 2–4 from control mice or periodontitis mice after the indicated manipulation of gene expression were seeded on 24-well plates at a concentration of 1 × 105 cells/well and incubated overnight before they were co-cultured with activated mouse CD4+ T cells. Mouse T lymphocytes were pre-activated in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 50 μM 2-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (all reagents were from Thermo Fisher Scientific) by plate-bound anti-CD3 antibody (1:1000 dilution; #ab16669; Abcam, USA) and soluble anti-CD28 antibody (1:5000 dilution; #ab283860, Abcam, USA) for 2–3 days in 24-well plates. Recombinant human TGF-β1 (2 ng/mL; R&D Systems, Minneapolis, MN, USA) and IL-2 (2 ng/mL; R&D Systems) were added into the culture medium to induce Treg differentiation; while Recombinant human TGF-β1 (2 ng/mL; R&D Systems) and IL-6 (50 ng/mL; R&D Systems) were added to induce Th17 differentiation. The activated mouse CD4+ T cells were loaded on the adherent BM-MSCs and co-cultured for further 2–3 days. The supernatant and T cells on top layers were collected for further analyses.
Recombinant human tgf β1
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany, China, Japan
Recombinant human TGF-β1 is a cytokine that regulates a variety of cellular processes, including cell growth, cell differentiation, and immune function. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (37 mentions)
Tgf β1, by R&D Systems (14 mentions)
Sb431542, by Merck Group (14 mentions)
Streptomycin, by Thermo Fisher Scientific (13 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (8 mentions)
E cadherin, by BD (8 mentions)
Vimentin, by Cell Signaling Technology (7 mentions)
β actin, by Cell Signaling Technology (7 mentions)
Snail, by Cell Signaling Technology (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Tgf β1, by Merck Group (7 mentions)
Rpmi 1640, by Thermo Fisher Scientific (6 mentions)
Smad2 3, by Cell Signaling Technology (6 mentions)
Hk 2 cell, by American Type Culture Collection (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
P smad2 3, by Cell Signaling Technology (6 mentions)
Smad3, by Cell Signaling Technology (6 mentions)
372 protocols using recombinant human tgf β1
1
Immunomodulatory effects of BM-MSCs on T cells
2
Culturing and Authentication of Cell Lines
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EPC2T and OKF6T were generated and cultured in keratinocyte-serum free medium (KSFM; Thermo Fisher Scientific, Waltham, MA, USA) as described previously.10 (link), 22 (link), 40 (link) TE11 cells were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) as previously described.40 (link) Countess™ Automated Cell Counter (Thermo Fisher Scientific) was used to count cells with 0.2% Trypan Blue dye to exclude dead cells. Recombinant human TGF-β1 (R&D Systems, Minneapolis, MN, USA) was reconstituted in Dulbecco’s Phosphate Buffered Saline (DPBS) containing 10% FBS. Chloroquine diphosphate solid salt (Sigma-Aldrich, St Louis, MO, USA) was reconstituted in water. CDDP (Sigma-Aldrich) and 5FU (Sigma-Aldrich) were dissolved in DMSO. Organotypic 3D culture (OTC) was carried out as described previously.19 (link) The earliest frozen stocks of all cell lines are stored at the Cell Culture Core of the University of Pennsylvania. We have propagated cells from frozen stocks of original vials that were authenticated by short tandem repeat profiling (ATCC) for highly polymorphic microsatellites to validate the identity of cells by comparing cells at the earliest stocks and those grown >8–12 passages. All cell lines undergo routine mycoplasma testing.
3
Culturing and Differentiating Cancer Cell Lines
4
Molecular Mechanisms of TGF-β1-Mediated Apoptosis
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA), emodin was obtained from Shanghai future industry Limited by Share Ltd (Shanghai, China) and BLM was acquired from Nippon Kayaku (Tokyo, Japan). The primary antibodies described in the study include: anti-E-cadherin, anti-vimentin, anti-cleaved caspase-3, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2, anti-Smad3, anti-phospho-Erk1/2 and anti-Erk1/2 (Cell Signaling Technology, CA, USA); anti-caspase-3, anti-Bax (Santa Cruz Biotechnology, CA, USA); anti-fibronectin (Proteintech, Chicago, USA); anti-caspase-8, anti-Bcl-2 (Absci, MD, USA); anti-TGF-β1, anti-FSP-1, anti-α-SMA (Abcam, USA); and anti-GAPDH (Beyotime Institute of Biotechnology, Haimen, China). Other reagents were obtained from Beyotime Institute of Biotechnology unless otherwise indicated.
5
Anthocyanidins Modulate Epithelial-Mesenchymal Transition
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Anthocyanidin compounds Cy (purity ≥ 96%), Dp, Pg, Mv (purity ≥ 97%) and Pt (purity ≥ 95%) were from Extrasynthese (Lyon, France). Recombinant human TGF-β1 was from R&D Systems (Minneapolis, MN). Electrophoresis reagents were from Bio-Rad (Mississauga, ON). The anti-Twist1 monoclonal antibody was from Santa Cruz Biotechnologies (Dallas, TX). The anti-Periostin antibody was from Abcam (Cambridge, MA). Antibodies against Snail, phospho-Smad2, Smad2, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), vimentin, β-Catenin, phospho-Fak, Fak, phospho-Akt and Akt were from Cell Signaling Technology (Beverly, MA). The anti-fibronectin antibody was from Sigma Aldrich (Oakville, ON). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-linked secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and enhanced chemiluminescence (ECL) reagents were from Denville Scientific Inc. (Metuchen, NJ). Micro bicinchoninic acid protein assay reagents were from ThermoFisher Scientific (Rockford, IL).
6
Knockdown Effects on Class Switch Recombination
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Class switch recombination was performed in CH12F3-2a cells as described previously50 (link). Briefly, RNF8, RNF168, L3MBTL2 or a combination of these, was knocked down using shRNAs. 40 hours later, cells were stimulated with ligands [1 ng/ml of recombinant human TGF-β1 (R&D Systems), 10 ng/ml of recombinant murine IL-4 (R&D Systems), and 250 ng/ml recombinant murine CD40 ligand (PerproTech)]. For analyzing class switch from IgM (IgM+/IgA−) to IgA (IgM−/IgA+), CH12F3-2 cells were collected after 60 hours, intracellularly stained with PE-conjugated anti-murine IgA antibody (clone 11–44-2, eBiosciences, Cat# 12-5994-82). Membrane IgM expression was assessed using FITC-conjugated anti-murine IgM antibody (eBiosciences; Cat# 11-5890-82). Cytofix/Cytoperm and Perm/Wash buffers (BD Biosciences, catalog # 554714) was utilized. Cells were then analyzed on a FACS Calibur (BD Biosciences) at the Mayo Clinic Flow Cytometry Core. Data was analyzed using FlowJo software (TreeStar).
7
Isolation and Stimulation of Human PBMCs
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Human peripheral blood mononuclear cells were isolated from healthy volunteers and stored at −80°C in cryovials at a 107-cell/ml concentration in FBS containing 10% DMSO. Before plating, cells were washed in PBS twice, recounted, and plated at a 106-cell/ml concentration in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine. Cells were stimulated for 3 days as described previously (12 (link)) with anti-human CD3 (BD no.555336, 0.3 µg/ml), anti-human CD28 (BD no.555725, 2 µg/ml) and recombinant human TGF-β1 (R&D no. 240B002, 2.5 ng/ml).
Bacteria isolated from human chloroform-resistant cultures were resuspended in PBS supplemented with protease inhibitor (Roche no. 4693159001) and phosphatase inhibitor (Roche no. 4906845001), heat-inactivated at 65°C for 1 h and sonicated for 10 min as described previously (14 (link)). Protein concentration in the resulting suspension was measured using the Pierce BCA protein assay kit (Thermo Scientific no. 23227). Bacterial extracts were added to PBMCs at 1 µg/ml 1 h after plating as described previously (13 (link)). PBS with the same protease inhibitor and phosphatase inhibitor was added as the no-bacterium control. Each human in vitro experiment contained at least 6 independent donor bacterial samples and was repeated at least twice.
Bacteria isolated from human chloroform-resistant cultures were resuspended in PBS supplemented with protease inhibitor (Roche no. 4693159001) and phosphatase inhibitor (Roche no. 4906845001), heat-inactivated at 65°C for 1 h and sonicated for 10 min as described previously (14 (link)). Protein concentration in the resulting suspension was measured using the Pierce BCA protein assay kit (Thermo Scientific no. 23227). Bacterial extracts were added to PBMCs at 1 µg/ml 1 h after plating as described previously (13 (link)). PBS with the same protease inhibitor and phosphatase inhibitor was added as the no-bacterium control. Each human in vitro experiment contained at least 6 independent donor bacterial samples and was repeated at least twice.
8
TGF-β1 Signaling Pathway Modulation
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The following reagents were obtained from the respective commercial vendors. Recombinant human TGF-β1 from R&D Systems (Minneapolis, MN, USA), losartan from Selleck Chemicals (Houston, TX, USA), dexamethasone from Sigma-Aldrich (St. Louis, MO, USA), ganciclovir from Selleck Chemicals, normal horse serum (RTU Vectastain Universal Elite ABC Kit) from Vector Laboratories (Burlingame, CA, USA), murine monoclonal anti-CMV IE antibody (anti-CMV Immediate Early Antigen Antibody, #LS-C103255) from LSBio (Seattle, WA, USA), a goat anti-mouse antibody (VectaFluor R.T.U. DyLight 488 anti-mouse) from Vector Laboratories, rhodamine phalloidin and Vecta-Stain mounting media (VECTASHIELD) from Invitrogen (Carlsbad, CA, USA), a TGF-β1 enzyme-linked immunosorbent assay (ELISA) kit (Total TGF-β1 ELISA Kit with precoated plates) from BioLegend (San Diego, CA, USA), and a MTS assay kit (CellTiter 96® AQueous One Solution Cell Proliferation Assay) from Promega (Medison, WI, USA).
9
Regulation of Myofibroblast Differentiation by CTGF
10
TGF-β Signaling Pathway Analysis
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Polyclonal anti-CTGF (diluted at 1:1000) and monoclonal anti-α-tubulin (diluted at 1:5000) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-Smad4 (diluted at 1:1000), anti-TGF-β receptor type I (diluted at 1:1000), anti-ERK1/2 (diluted at 1:2000), monoclonal anti-Smad2 (diluted at 1:1000) and anti-Smad3 (diluted at 1:1000) antibodies were obtained from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (diluted at 1:5000) were obtained from Bio-Rad Laboratories (Hercules, CA), and horseradish peroxidase-conjugated donkey anti-goat IgG (diluted at 1:5000) was obtained from Santa Cruz Biotechnology. Recombinant human TGF-β 1 was obtained from R&D systems (Minneapolis, MN). SB431542 was obtained from Sigma-Aldrich (Oakville, ON), and U0126 was obtained from Calbiochem (San Diego, CA).
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