Sphere cells were used after six days of culture in serum-free modified medium. On the day of transfection, sphere cells were washed once with serum-free growth medium without antibiotics and cells were seeded at a density of 2–3 × 106 cells per well in 0.8 ml of serum-free growth medium without antibiotics. Lipofectamine® LTX & Plus Reagent (Catalog no-15338-100, Invitrogen, CA, USA) was used according to the manufacturer’s instructions for the transfection assay. The plasmid vectors used for the transfection assay were pGFP.TGFRBKDEL and empty pCMV control vector (obtained from Dr. B.K. Ambati, Moran Eye Center, UT, USA). The TGFβ-KDEL vector interferes with TGFβ signaling through anomalous sorting of cytokine bound to the expressed altered receptor (Singh et al., 2011 (link)). α-SMA was analyzed at six days after transfection and culture of cells in presence or absence of 1.0 ng/ml TGFβ (T7039, Sigma, St. Louis, MO, USA) with 2.0 ng/ml PDGF-AA or PDGF-BB (CYT-590 or CYT-590, ProSpec-Tany Technogene Ltd., PO Box 6591, East Brunswick, NJ).
T7039
Manufactured by Merck Group
Sourced in United States
T7039 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of T7039 is to provide a controlled environment for scientific experiments and procedures. Further details about the product's specific features or intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Tgf β1, by Merck Group (2 mentions)
Lipofectamine ltx plus reagent, by Thermo Fisher Scientific (1 mentions)
M3763, by Merck Group (1 mentions)
S4317, by Merck Group (1 mentions)
A9251, by Merck Group (1 mentions)
Plasminogen, by Merck Group (1 mentions)
Verteporfin, by Merck Group (1 mentions)
Ab2413, by Santa Cruz Biotechnology (1 mentions)
D val leu lys 7 amido 4 methylcoumarin, by Merck Group (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab14715, by Abcam (1 mentions)
Ab66705, by Abcam (1 mentions)
Sr11302, by Bio-Techne (1 mentions)
Pd184352, by Bio-Techne (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
L3129, by Merck Group (1 mentions)
Tgfβ1 t7039, by Merck Group (1 mentions)
Ab133145, by Abcam (1 mentions)
Anti il 23 antibody, by Thermo Fisher Scientific (1 mentions)
Anti il 17a, by Thermo Fisher Scientific (1 mentions)
7 protocols using t7039
1
Transfection of Sphere Cells to Modulate TGF-β Signaling
2
Modulating CD73 and TGF-β Pathways
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3
Investigating PAI-1 and Fibronectin Regulation
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Verteporfin, human transforming growth factor (TGF-β1; T7039), plasminogen and D-Val-Leu-Lys-7-amido-4-methylcoumarin were from SigmaAldrich. OXA-06, PD-184352, SR-11302 were from Tocris. For protein detection specific antibodies against PAI-1 (Abcam Cat# ab66705, RRID:AB_1310540), fibronectin (abcam, ab2413), p-ERK-1/2 (Santa Cruz, E4, sc-7383), SDHA (abcam, ab14715) and histone H3 (abcam, ab1791) were used.
4
Fibroblast and Epithelial Cell Response to TGF-β1
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Endometrial tissue samples (n = 11; category I of Kenney and Doig1 at the early luteal phase of the estrous cycle) were used. When fibroblast (n = 6) and epithelial cell (n = 5) cultures from passage 1 reached confluence, the culture medium was replaced with fresh Dulbecco’s Modified Eagle Medium (DMEM)/Ham’s F-12 supplemented with gentamicin (20 μg/ml) and BSA (0.1%; w/v) at 38.0 °C in an atmosphere of 5% CO2 in air. Fibroblasts and epithelial cells were always treated with vehicle or TGF-β1 (5 ng/ml; T7039; Sigma-Aldrich) for 24 h and 48 h. The dose of TGF-β1 was chosen in a preliminary study. After treatment with TGF-β1, conditioned medium was collected for MMP and TIMP determination using ELISA and MMP-2/9 gelatinolytic activity using zymography. The cells were harvested into TRIZOL Reagent (no. 15596-026; Invitrogen, Carlsbad, CA) and kept at −80 °C until RNA extraction, reverse transcription, and real-time PCR were performed. To normalize MMP and TIMP concentrations, the protein concentration was quantified.
5
Stat5 and GLRX Regulate TGFβ1 Signaling
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293T cells were cotransfected with the pGL3-Glrx or pGL3-mutant Glrx, and pcDNA3.1+-Stat5a or pCDH-Stat5b for 24 hours. LX2 and 293T cells were transfected with the Smad3-responsive luciferase reporter SBE-Luc (a gift from X. Cao, Johns Hopkins School of Medicine), together with Smad3- and/or GLRX-expressing plasmids for 24 hours in the absence or presence of TGFβ1 (5 ng/ml) (T7039, Sigma-Aldrich) stimulation. Luciferase activity was measured and normalized for transfection efficiency against β-galactosidase activity from the cotransfected pCMX-β-gal (44 (link)).
6
Isolation and Stimulation of Murine Macrophages
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BMDMs and SPMs were primarily cultured for in vitro experiments. In detail, bone cavity of the hind legs and the femurs obtained from the hip joint were flushed using a 10 ml syringe filled with DMEM attached to BD 26 ½” G needle (#21000578, BD PrecisionGlide). Spleen was extracted from the murine abdominal cavity and was crushed onto a 40 μm sterile filter with DMEM. After centrifuge, cell pellet was resuspended and cultured in macrophage medium (DMEM + 20% L929 Conditional Media +10% FBS + 1% Penicillin–Streptomycin) in 37 °C, 5% CO2 incubator. On day 7, macrophages were obtained by non-enzymatic cell dissociation from the 75-cm2 flask for experiments. Both BMDMs and SPMs were stimulated with LPS 100 ng/ml (#L3129, Sigma-Aldrich) + IFNγ 10 ng/ml (#575306, Biolegend), or IL-4 10 ng/ml (#574304, Biolegend) + IL-13 10 ng/ml (#575902, Biolegend) or TGFβ1 5 ng/ml (#T7039, Sigma-Aldrich) for 4 hrs, 8 hrs, and 24 hrs, as indicated.
7
Macrophage-derived factors modulate Th17 and Treg differentiation
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Primary macrophages were cultured for 24 h, as aforementioned. The media were collected and filtered using 0.22 μm filters and was defined as CM. Purified CD4+ naive T cells were cultured under Th17 cell-polarizing conditions (2 ng/mL TGF-β1, T7039, Sigma-Aldrich) or Treg-polarizing conditions (10 ng/mL IL-2, SRP6170, Sigma-Aldrich; 20 ng/mL TGF-β1, T7039, Sigma-Aldrich), combined with or without macrophages-CM (normal medium: CM = 2:1). For recombinant human FABP4 (rhFABP4) treatment, 10 μg/mL rhFABP4 (ab133145, Abcam) was added in the culture system. For neutralization experiments, 10 μg/mL anti-IL-17A (16-7178-81, eBioscience, Thermo Fisher Scientific) or anti-IL-23 antibody (16-7222-82, eBioscience) was added in the culture system
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