C2C12 cells were obtained from the American Type Culture Collection (CRL-1772: Manassas, VA, USA) and cultured in Dulbecco's modified minimum essential medium (DMEM) supplemented with 20% fetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin:streptomycin solution (50U/mL and 50 µg/mL, respectively; Gibco) at 37°C in air with a humidified atmosphere of 5% CO2. An AVA stock solution was made in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the cultured medium was 0.05%. A total of 30 μmol synthetic AVAs (AVA-A, -B, and -C) provided by Dr. Mitchell Wise (USDA Cereal Research Laboratory, Madison, WI, USA) were treated with fresh medium, when the cells reached 70% confluence. An equivalent amount of DMSO was added to the control cells. After the AVA was treated for 24 h, recombinant mouse TNF-α (Roche, Basel, Switzerland) was added to a final concentration of 10ng/mL for various incubation times depending on the experimental protocols.
Recombinant mouse tnf α
Manufactured by Roche
Sourced in Switzerland, United States
Recombinant mouse TNF-α is a laboratory reagent used for research purposes. It is a cytokine produced from recombinant DNA technology. This product is intended for in vitro use only.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
C2c12 cell, by American Type Culture Collection (1 mentions)
Acetylcholine chloride, by Merck Group (1 mentions)
Wortmannin, by Cayman Chemical (1 mentions)
Bapta am, by Thermo Fisher Scientific (1 mentions)
Nω nitro l arginine methyl ester hydrochloride l name, by Merck Group (1 mentions)
Forskolin, by Cayman Chemical (1 mentions)
Phorbol 12 myristate 13 acetate pma, by Cayman Chemical (1 mentions)
U0126, by Cayman Chemical (1 mentions)
Gsk1016790a gsk101, by Merck Group (1 mentions)
Sto 609, by Cayman Chemical (1 mentions)
R phenylephrine hydrochloride pe, by Merck Group (1 mentions)
Aicar, by Cayman Chemical (1 mentions)
Sodium nitroprusside snp, by Merck Group (1 mentions)
Compound c, by Cayman Chemical (1 mentions)
Ly294002, by Cayman Chemical (1 mentions)
Taurine, by Merck Group (1 mentions)
3 protocols using recombinant mouse tnf α
1
Investigating AVAs and TNF-α in C2C12 Cells
2
Calcium Signaling Modulators Protocol
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TRPV4 specific agonist GSK1016790A (GSK101, #G0798) and inhibitor GSK2193874 (GSK874, #5106) were obtained from Sigma-Aldrich (St. Louis, MO) and Tocris Bioscience (Bristol, UK), respectively. Compound C (AMPK inhibitor, #11967), STO-609 (calmodulin-dependent kinase kinase (CaMKK) inhibitor, #15325), Wortmannin (PI3K inhibitor, #10010591), LY294002 (Akt inhibitor, #70920), U0126 (ERK1/2 inhibitor, #70970), AICAR (#10010241), Forskolin (#11018), and Phorbol 12-myristate 13-acetate (PMA, #10008014) were obtained from Cayman Chemical (Ann Arbor, MI). Ruthenium Red (Ca2+ fluxes blocker, #557450), and H-89 (PKA inhibitor, #371963) were obtained from Calbiochem (San Diego, CA). Calcium chelator BAPTA/AM (#B-1205) was from Invitrogen (Grand Island, NY). Recombinant mouse TNF-α was from Roche (#11271156001, Indianapolis, IN). Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, #N5751), Sodium nitroprusside (SNP, #PHR1423), Acetylcholine chloride (#A2661), (R)-(−)-Phenylephrine hydrochloride (PE, #P8155) and other chemicals were obtained from Sigma-Aldrich.
3
Rat Myogenic Cells Differentiation
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L6 rat myogenic cells were seeded at the density of 25,000/cm2 and cultured in growth medium (GM) consisting of: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide and 95% air. Twenty-four hours after plating, cultures were washed with Phosphate Buffer Saline (PBS) and shifted to low-serum medium consisting of DMEM supplemented with 1% fatty acid-free bovine serum albumin (BSA, #A9418 Sigma, St. Louis, MO, USA) [42 (link)] and treated with 200 μM Taurine (#T8691 Sigma, St. Louis, MO, USA) and/or 15 ng/mL recombinant mouse TNF-α (#11271156001; Roche, Indianapolis, IN, USA) at different times as described below.
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