NOS2 cells were stimulated with recombinant human transforming growth factor-β1 (TGF-β1) (Wako Pure Chemical Industries, Osaka, Japan) at 10 ng/ml in RPMI-1640 supplemented with 2% FBS for 96 h. After a 96-h incubation with TGF-β1, the cells were washed with PBS and used for a number of assays.
Tgf β1
Manufactured by Fujifilm
Sourced in Japan, United States
TGF-β1 is a laboratory reagent used for research purposes. It is a protein that plays a crucial role in various cellular processes, including cell growth, differentiation, and immune function. The core function of TGF-β1 is to serve as a signaling molecule that helps regulate the behavior of cells in in vitro studies.
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Lab products found in correlation
Activin a, by Fujifilm (2 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Rpmi 1640, by Nissui Pharmaceutical (1 mentions)
Fetal bovine serum (fbs), by Nissui Pharmaceutical (1 mentions)
Dexamethasone (dex), by Fujifilm (1 mentions)
β glycerophosphate, by Fujifilm (1 mentions)
Igf 1, by Fujifilm (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Fujifilm (1 mentions)
Aphidicolin, by Fujifilm (1 mentions)
Oris 96 well cell migration assay kit, by Platypus Technologies (1 mentions)
Calcein am, by Dojindo Laboratories (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
Insulin like growth factor 1 igf 1, by Merck Group (1 mentions)
Bmp 2, by Merck Group (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Fujifilm (1 mentions)
Dmem low glucose, by Fujifilm (1 mentions)
Tgf β1, by Merck Group (1 mentions)
10 protocols using tgf β1
1
TGF-β1 Induced NOS2 Cell Stimulation
2
Collagen Synthesis in Lung Fibroblasts
3
Osteogenic Differentiation of Human iPSCs
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We previously reported an effective method for inducing osteogenesis in human iPSCs [21 (link),22 (link)]. Briefly, human iPSCs were incubated in the presence or absence of a SMO agonist or inhibitor in osteoblast differentiation medium (OBM). The OBM consisted of α-MEM (Invitrogen) supplemented with 10% FBS, 50 μg/ml L-ascorbic acid (Wako Pure Chemical Industries Ltd.), 10 mM β-glycerophosphate (Wako Pure Chemical Industries Ltd.), and 10 nM dexamethasone (Wako Pure Chemical Industries Ltd.). A combination of cytokines, referred to as FIT and comprising 25 ng/ml bFGF, 1 ng/ml TGF-β1 (Wako Pure Chemical Industries Ltd.), and 100 ng/ml IGF-1 (Wako Pure Chemical Industries Ltd.), was added on the following day (day 0). The iPSCs were cultured for an additional 10 days, with the OBM replenished every 3 days.
4
Cell Migration Assay with TGF-β1
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MIO-M1 cell migration was evaluated using an Oris 96-well cell migration assay kit (Platypus Technologies, Madison, WI), according to the manufacturer’s instructions. Cells were seeded in each well and transfected with siRNAs. After 1 hour of pretreatment with 1% FBS/DMEM containing 10 ng/ml TGF-β1 with 5 mM aphidicolin (Fuji Film Wako Pure Chemicals, Osaka, Japan) to inhibit cell division, the stoppers were removed to allow cells to migrate into the detection zone. The cells were then incubated for 48 hours and stained with PBS containing calcein AM (Dojindo, Kumamoto, Japan) for 1 hour. The signal intensity of the stained cells that migrated into the detection zone was measured with an Infinite F200 PRO microplate reader in the fluorescence mode (TECAN, Männedorf, Switzerland) using a fluorescence filter set (excitation, 485 nm; emission, 535 nm) with an Oris detection mask attached to the bottom of the plate.
5
Muse Cell Differentiation Protocol
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Muse cells were plated at 1.55 × 104/cm2 and cultured in the adherent state for the entire period of induction on a laminin-coated surface. The cells were first incubated with DMEM low glucose containing 2% of FBS plus TGF-β1 (2.5 ng/mL, Wako), BMP-4 (5 ng/mL, Wako), BMP-2 (5 ng/mL, Sigma-Aldrich), activin A (10 ng/mL, Wako), Wnt-3a (50 ng/mL, R&D Systems, Minneapolis, MN, USA), and bFGF (10 ng/mL, Wako) for 7 d and then for 2 wk with DMEM low glucose containing 2% of FBS plus TGF-β1, insulin-like growth factor-1 (IGF-1, 5 ng/mL, Sigma-Aldrich), hepatocyte growth factor (HGF; 20 ng/mL, Wako), and cardiotrophin-1 (CT-1; 200 ng/mL, Sigma-Aldrich).
6
Cytokine-induced HEK293 and NHDF cell culture
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Human embryonic kidney 293 (HEK293) (KAC Co, Ltd (Kyoto, Japan)) and HEK293T cells were cultured in Eagle’s minimum essential medium (FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 1 × nonessential amino acids (FUJIFILM Wako Pure Chemical) and 10% (v/v) fetal bovine serum (FBS). HEK293 cells were seeded at a density of 7.5 × 105 cells/well in a 24-well plate and incubated with or without IL-1β (1 ng/ml) and TGF-β1 (10 ng/ml) (FUJIFILM Wako Pure Chemical).
Normal human dermal fibroblasts (NHDFs), Detroit 551, were obtained from the American Type Culture Collection (VA, USA). The Detroit 551 cells were maintained in Eagle’s minimum essential medium supplemented with 1 × nonessential amino acids, 1 mM sodium pyruvate, and 10% (v/v) FBS. The Detroit 551 cells were seeded at a density of 2.5 × 104 cells/well in a 24-well plate and incubated with or without a mixture of the cytokines (0.1–1.0 ng/ml, TNF-α, IL-1β, and IL-6), IL-1β (1.0 ng/ml) or TGF-β (10 ng/ml) (FUJIFILM Wako Pure Chemical).
Normal human dermal fibroblasts (NHDFs), Detroit 551, were obtained from the American Type Culture Collection (VA, USA). The Detroit 551 cells were maintained in Eagle’s minimum essential medium supplemented with 1 × nonessential amino acids, 1 mM sodium pyruvate, and 10% (v/v) FBS. The Detroit 551 cells were seeded at a density of 2.5 × 104 cells/well in a 24-well plate and incubated with or without a mixture of the cytokines (0.1–1.0 ng/ml, TNF-α, IL-1β, and IL-6), IL-1β (1.0 ng/ml) or TGF-β (10 ng/ml) (FUJIFILM Wako Pure Chemical).
7
VASH2 Knockdown in DISS and SKOV3 Cells
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DISS and SKOV3 cells and VASH2 knocked‐down (sh‐VASH2) clonal cell lines from DISS cells were described previously.21 Cells were maintained in DMEM (Wako, Saitama, Japan) with 10% FBS (Sigma‐Aldrich) and penicillin–streptomycin (Wako). For experimental treatments, the serum was reduced to 0.5% and 10 ng/mL TGF‐β1 (Wako) or 5 μM SB431542 (Wako) was added.
8
TGF-β1 and FGF2 Modulate EMT in RLE Cells
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Rat AEC2 cell line RLE-6TN (RLE) cells [48 (link)] were obtained from American Type Culture Collection (ATCC). RLE cells were cultured in Dulbecco’s-modified Eagle’s medium/Nutrient F-12 Ham (DMEM/F12) (Sigma) containing 10% FBS and subcultured every 2 d. To induce EMT, 1 104 cells/cm2 were cultured in DMEM/F12 containing 1% FBS for 24 h and then stimulated with 0.5 ng/ml recombinant human TGF-β1 (R&D Systems) dissolved in 4 mM HCl containing 0.1% bovine serum albumin for 48 h. To assess the effect of FGF2 on EMT, cells were stimulated with 0.5 ng/ml TGF-β1 and 10–100 ng/ml recombinant human FGF2 (Wako) with 100 μg/ml heparin. To analyze signaling pathways, cells were pretreated with 2–10 μM U0126 (Promega) or 10 μM LY294002 (Promega) for 30 min and then stimulated with 0.5 ng/ml TGF-β1 alone or in combination with 50 ng/ml FGF2.
9
Isolation and Culture of Rheumatoid Arthritis Synovial Cells
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A total of 6 patients with RA diagnosed based on the revised 1987 American College of Rheumatology criteria for RA were recruited. Synovial tissues were collected from 6 patients who underwent arthroplastic joint surgery and synovectomy.
Synovia obtained from RA patients were aseptically dissected from the surrounding tissues, minced, and enzymatically digested with 2 mg/mL Clostridium collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 5-10 μg/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO, USA) for 2-3 h. After digestion, the resulting single cell suspension was washed, filtered through sterile gauze and nylon mesh, washed thoroughly again, and finally resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum. The cells were then cultured overnight to allow the cells to adhere to the culture plate. After washing the plate to remove nonadherent cells, the remaining adherent cells were cultured with the indicated cytokines, and adherent cells of 2-3 passages were further used in this study. The recombinant human cytokines used in this study were as follows: IL-1β, TNFα, IL-6, IL-6Rα, IL-17A, M-CSF (all BioLegend, San Diego, CA, USA), TGF-β 1 , activin A, and RANKL (all Wako Pure Chemical Industries).
Synovia obtained from RA patients were aseptically dissected from the surrounding tissues, minced, and enzymatically digested with 2 mg/mL Clostridium collagenase (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and 5-10 μg/mL deoxyribonuclease 1 (Sigma Chemical Co., St. Louis, MO, USA) for 2-3 h. After digestion, the resulting single cell suspension was washed, filtered through sterile gauze and nylon mesh, washed thoroughly again, and finally resuspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum. The cells were then cultured overnight to allow the cells to adhere to the culture plate. After washing the plate to remove nonadherent cells, the remaining adherent cells were cultured with the indicated cytokines, and adherent cells of 2-3 passages were further used in this study. The recombinant human cytokines used in this study were as follows: IL-1β, TNFα, IL-6, IL-6Rα, IL-17A, M-CSF (all BioLegend, San Diego, CA, USA), TGF-β 1 , activin A, and RANKL (all Wako Pure Chemical Industries).
10
Differentiation of TH1 and TH17 Cells
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For the differentiation of TH1 cells35 (link), CD45.1+OT-II CD4+ T cells (2×105) were cultured with pDCs (2×104) in the presence or absence of IMQ (5 μg/ml) in combination with OVA323-339 peptide (1 μM), anti-IL-4 mAb (10 μg/ml; 11B11, BD Biosciences) and recombinant mouse IL-2 (0.2 ng/ml; Wako Pure Chemicals) for 3 days in 96-well flat-bottomed plates. For the differentiation of TH17 cells35 (link), CD45.1+OT-II CD4+ T cells (2×105) were cultured with pDCs (2×104) in the presence or absence of IMQ (5 μg/ml) in combination with OVA323–339 peptide (1 μM), anti-IFN-γ mAb (10 μg/ml; R4-6A2, BD Biosciences), anti-IL-4 mAb (10 μg/ml; 11B11), recombinant mouse IL-2 (0.2 ng/ml) and recombinant human transforming growth factor (TGF)-β1 (10 ng/ml; Wako Pure Chemicals) for 3 days in 96-well flat-bottomed plates. In some experiments, anti-IL-6 mAb (10 μg/ml; MP5-20F3, eBioscience), anti-IL-12 mAb (10 μg/ml; C17.8, eBioscience) or anti-IFNAR1 mAb (10 μg/ml; MAR1-5A3, eBioscience) was added to the culture. Analysis of the expression of IFN-γβ and/or IL-17 among gated CD4+ T cells was performed by flow cytometry as described above.
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