Trimethylamine N-oxide (Sigma-Aldrich, Saint Louis, MO, USA) solubilized in culture medium or Tyrode standard solution according to the protocol used, was freshly prepared for each experiment. Menadione (MEN) (Sigma-Aldrich) was solubilized in DMSO at an initial concentration of 100 mM and then diluted in culture medium to the final concentration of 100 μM. ATP was prepared in Tyrode standard solution at a concentration of 10 mM and diluted in culture medium to the final concentration of 100 μM. Tyrode standard solution used in different experiments contained (in mM): 154 NaCl, 4 KCl, 1 MgCl2, 5.5 D-glucose, 5 HEPES, 2 CaCl2, pH adjusted to 7.34 with NaOH. Unless otherwise specified, all reagents for cell culture and experiments were purchased from Sigma-Aldrich.
Menadione men
Manufactured by Merck Group
Sourced in Italy
Menadione (MEN) is a chemical compound that serves as an essential nutrient and a precursor to vitamin K. It is a synthetic form of vitamin K3 and is commonly used in laboratory settings for various research and analytical applications. Menadione's core function is to provide a source of vitamin K activity, which is crucial for various physiological processes, including blood coagulation and bone metabolism.
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Lab products found in correlation
Trimethylamine n oxide, by Merck Group (1 mentions)
Potato dextrose agar (pda), by Merck Group (1 mentions)
Coumarin 6 cu6, by Merck Group (1 mentions)
Mops acid, by Merck Group (1 mentions)
Rpmi medium, by Merck Group (1 mentions)
L cys, by Merck Group (1 mentions)
L gsh, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Halofuginone, by Cayman Chemical (1 mentions)
Tunicamycin tm, by Merck Group (1 mentions)
Sodium arsenite, by Merck Group (1 mentions)
Thapsigargin tg, by Santa Cruz Biotechnology (1 mentions)
Ire1α inhibitor 4μ8c, by Bio-Techne (1 mentions)
Perk inhibitor gsk2606414, by Merck Group (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using menadione men
1
Preparation of TMAO and Menadione Solutions
2
Pterostilbene-loaded PLGA Nanoparticles
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Pterostilbene was purchased from Chemodex (St. Gallen, Switzerland). Poly(d ,l )-lactic-co-glycolic acid (PLGA, lactide: glycolide 50:50, MW 50 kDa), coumarin 6 (Cu6) (98%), potato dextrose agar (PDA), fluopyram, RPMI medium (RPMI 1640 with l -glutamine, without bicarbonate), MOPS acid, XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-(carbonyl (phenylamino)]-2H-tetrazolium hydroxide] and menadione (MEN) were purchased from Sigma-Aldrich (Milan, Italy).
Potato dextrose broth (PDB) was purchased from Formedium LTD (Hunstanton, Norfolk, England).
The microfluidic flow focusing reactor was assembled by the research group involved in the study as reported previously by Bramosanti et al.23 (link).
Potato dextrose broth (PDB) was purchased from Formedium LTD (Hunstanton, Norfolk, England).
The microfluidic flow focusing reactor was assembled by the research group involved in the study as reported previously by Bramosanti et al.23 (link).
3
Metabolic Engineering of Streptomyces Strains
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LE-overproducing Streptomyces globisporus 1912 strain was obtained in the laboratory of B. Matselyukh (D.K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv). LE (99.5% purity, according to HPLC data) was prepared in the laboratory of J. Rohr (University of Kentucky, USA) and dissolved in absolute ethanol to obtain a 4 mg/ml stock solution. LA was isolated from S. cyanogenus S-136 following a previously published procedure, and L was prepared by hydrolysis using formic acid39 ,40 .
Menadione (MEN, Sigma Aldrich) was diluted in DMSO to obtain a 10 mM stock and stored at −20 °C. The thiol containing substances glutathione (L-GSH, ≥ 98%, Sigma Aldrich) and cysteine (L-Cys, ≥97%, Sigma Aldrich) and, in addition, the chemical GSH precursor N-acetylcysteine (NAC, ≥ 99%, Sigma Aldrich) were prior to each experiment freshly diluted in the indicated buffer or growth medium at indicated concentrations. GSH-synthesis inhibitor BSO (≥ 97%, Sigma Aldrich) was freshly diluted in ddH2O prior to each experiment. All the other chemicals and solvents were purchased from Sigma-Aldrich and used without further purification.
Menadione (MEN, Sigma Aldrich) was diluted in DMSO to obtain a 10 mM stock and stored at −20 °C. The thiol containing substances glutathione (L-GSH, ≥ 98%, Sigma Aldrich) and cysteine (L-Cys, ≥97%, Sigma Aldrich) and, in addition, the chemical GSH precursor N-acetylcysteine (NAC, ≥ 99%, Sigma Aldrich) were prior to each experiment freshly diluted in the indicated buffer or growth medium at indicated concentrations. GSH-synthesis inhibitor BSO (≥ 97%, Sigma Aldrich) was freshly diluted in ddH2O prior to each experiment. All the other chemicals and solvents were purchased from Sigma-Aldrich and used without further purification.
4
Induction of ER Stress and ISR Pathways
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To induce ER stress, cells were treated with 5 μg/ml tunicamycin (TM; Sigma‐Aldrich) or 1 μM thapsigargin (TG; Santa Cruz Biotechnology) for indicated times. HRI activation was triggered by 5 μM sodium arsenite (Sigma‐Aldrich) treatment for 20 h, PKR activation by 20 μM BEPP (Sigma‐Aldrich) treatment for 6 h and GCN2 activation by 10 nM Halofuginone (Cayman Chemical Company) treatment for 20 h, and ROS was generated by 100 μM menadione (Men; Sigma‐Aldrich) treatment for 4 h. Treatment with 0.5 μM PERK inhibitor GSK2606414 (Sigma‐Aldrich), 40 μM IRE1α inhibitor 4μ8C (Tocris), 75 μM angiogenin inhibitor NSC‐65828 [8‐amino‐5‐(4′‐hydroxybiphenyl‐4ylazo)naphthalene‐2‐sulfonate] (National Cancer Institute (NCI)) and 5 μM mTOR inhibitor Torin1 (Tocris) was started 1 h before induction of ER stress. Treatment with 200 nM ISR inhibitor ISRIB (Selleck Chemicals) was started 2 h before fixation, and treatment with 5 μM HRI inhibitor hemin chloride (Santa Cruz Biotechnology) was started 3 h before fixation. Compounds were dissolved in H2O (sodium arsenite), 100 mM NaOH (hemin chloride) or DMSO (rest). For all treatments, equal volumes of solvents were added to the controls. To determine ROS levels, cells were incubated with 5 μM CellROX green reagent (Thermo Fisher) 30 min before fixation.
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