Proliferation of ASCs was measured using the xCelligence real-time cell analyzer system from Roche (Roche Applied Science, Penzberg, Germany, http://www.roche.com ). In brief, 800 cells/well of NT, LV-CTRL, LV#3, and LV#6 mASCs or NT, LV-CTRL, LV#18, and LV#19 hASCs were added to 16-well E-plates as described previously 30 (link). The E-plates were then placed on the device station in the incubator (normoxia; 5% CO2 at 37°C) for continuous recording of impedance, as reflected by cell index. In some experiments, a TGF-β1/2/3-neutralizing antibody (1D11; R&D Systems, Minneapolis, MN, http://www.rndsystems.com ) or SB431542 (Sigma) was added on days 1 and 3 to the wells at 2.5 µg/ml and 10 µM, respectively. Cell proliferation was also assayed using the CellTiter-Blue reagent, according to the manufacturer's instructions (Promega, Madison, WI, http://www.promega.com ). In brief, NT, LV-CTRL, LV#3, and LV#6 mASCs were seeded in 96-well plates (800 cells/well) and cultured at 5% O2/5% CO2 at 37°C. On days 1, 3, and 7, CellTiter-Blue was added to the cells during the last 4 hours of culture and fluorescence was measured at 560 nm on a Glomax multidetection system (Promega).
Tgf β1 2 3 neutralizing antibody 1d11
Manufactured by R&D Systems
The TGF-β1/2/3 neutralizing antibody 1D11 is a laboratory tool used to neutralize the activity of transforming growth factor beta 1, 2, and 3 proteins. This antibody can be utilized in various in vitro and ex vivo experiments to study the biological functions of the TGF-β family of proteins.
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Lab products found in correlation
Horse serum, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by R&D Systems (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
C2c12 cell, by American Type Culture Collection (2 mentions)
Activin a, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Xcelligence real time cell analyzer system, by Roche (1 mentions)
Sb431542, by Merck Group (1 mentions)
Celltiter blue reagent, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
3 protocols using tgf β1 2 3 neutralizing antibody 1d11
1
Real-time Cell Proliferation Assay for ASCs
2
C2C12 Myogenic Differentiation Assay
3
C2C12 Cell Differentiation and Growth Factor Treatments
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C2C12 cells (ATCC, Manassas, VA) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO). Differentiation was induced when cells reached approximately 80% confluence by switching to differentiation medium, DMEM supplemented with 2% horse serum (Life Technologies). Cells were treated with growth factors or inhibitors after three days of differentiation. For treatment with the inhibitors ActRIIB-Fc (generously provided by Qian Wang, Stony Brook University Medical Center, produced as previously described [27 ]) and TGF-β1/2/3 neutralizing antibody 1D11 (R&D Systems, Minneapolis, MN), media was changed to DMEM supplemented with 0.1% bovine serum albumin 24 hours before the addition of the inhibitors. For treatment with recombinant myostatin, activin A, TGF-β1 and insulin-like growth factor 1 (IGF-1) (all obtained from R&D Systems), media was changed to DMEM supplemented with 0.1% bovine serum albumin and the indicated growth factor on the third day post differentiation media change. For all treatments, cells were incubated for 5 hours and harvested in TRIzol (Life Technologies) for qRT-PCR analysis.
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