Polysorbate 20 (Acros, >95%), sodium dodecyl sulfate (SDS; Fisher, ≥ 99%), 1-butanol (Fisher, 99.98%), toluene (Fisher, 99.9%), potassium nitrate (Fisher, ≤ 3ppm heavy metals, iron), ferrocene (Alfa Aesar, 99%), and menadione (MP Biomedicals, ≥ 98%) were used as received. Chemical structures of surfactants and electroactive species are shown in Figure 1 .
Menadione
Manufactured by MP Biomedicals
Sourced in United States
Menadione is a synthetic form of vitamin K. It is commonly used as a chemical reagent in various laboratory applications, including biological and biochemical research.
Automatically generated - may contain errors
Lab products found in correlation
Hemin, by Thermo Fisher Scientific (2 mentions)
Yeast extract, by Merck Group (2 mentions)
Bhi broth, by Merck Group (2 mentions)
L cysteine, by Merck Group (2 mentions)
Polysorbate 20, by Thermo Fisher Scientific (1 mentions)
Toluene, by Thermo Fisher Scientific (1 mentions)
Potassium nitrate, by Thermo Fisher Scientific (1 mentions)
1 butanol, by Thermo Fisher Scientific (1 mentions)
Ferrocene, by Thermo Fisher Scientific (1 mentions)
Filmtracer sypro ruby biofilm matrix stain, by Thermo Fisher Scientific (1 mentions)
6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Menadione, by Thermo Fisher Scientific (1 mentions)
Cellrox green, by Thermo Fisher Scientific (1 mentions)
Necrosulfonamide nsa, by Bio-Techne (1 mentions)
Gsk 872, by Merck Group (1 mentions)
Necrostatin 1s nec 1s, by Merck Group (1 mentions)
Fk866, by Selleck Chemicals (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Nicotinamide, by Merck Group (1 mentions)
α amanitin, by Cayman Chemical (1 mentions)
12 protocols using menadione
1
Electrochemical Detection of Menadione
2
Antifungal Activity Evaluation Protocol
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All materials were obtained from Sigma Aldrich (Gillingham, UK) unless otherwise stated. All solvents were obtained from Fisher Scientific, Loughborough, UK. Amphotericin B (AmB) was obtained from Cayman Chemical (Cambridge Bioscience), Cambridge, UK. Candida species were obtained from Thermo Scientific, Loughborough, UK. Menadione was obtained from MP Biomedicals, Irvine, CA, USA. Texas Red succinimidyl ester, Concanavalin A Alexa Flour™ 488 Conjugate, Film Tracer™ SYPRO™ Ruby Biofilm Matrix Stain, and 6-diamidino-2-phenylindole (DAPI) were all obtained from Molecular Probes, Loughborough, UK.
3
Quantifying ROS in Lung Cells
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Beas-2B or pSAECs were plated with 5000 and 7500 cells per well, respectively, in clear-bottom, black-walled 96-well microplates and cultured 2 days prior to treatment with incinerated thermoplastics. At designated time points, thermoplastic- and menadione-treated cells were stained with 5 μM of CellROX Green (ThermoFisher Scientific) and 1 μM Hoechst 33342 in complete medium for 30 min under standard culture conditions and visualized using the ImageXpress. Nuclei were visualized using a standard DAPI filter set, while CellROX was visualized using a FITC filter set. Treatment with 100 μM menadione (MP Biomedicals, LLC.; Solon, OH) for 60 min in complete growth medium was used as a positive ROS control prior to ROS assessment. After measurement/imaging, cells were fixed using 4% formaldehyde for 15 min at room temperature. A binary mask was generated based on nuclear Hoechst 33342 intensity from the DAPI channel and overlaid onto each nucleus for single cell identification. An intensity-based mask for the FITC channel was applied covering the nucleus and cytoplasm using the MetaXpress. Cell-specific masks were then quantitated for average intensity prior to comparisons.
4
Macrophage Viability Assays with Compounds
5
Anaerobic Biofilm Cultivation in Oxic Conditions
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A modified brain heart infusion (M-BHI) broth was used for cultivations. BHI broth (Sigma Aldrich, USA) was supplemented with 5 mg/L hemin (Alpha Aesar, USA), 1 mg/L menadione (MP Biomedicals, LLC, France), 0.1 g/L L-cysteine (Sigma, USA) and 1 g/L yeast extract (Sigma, USA). The growth media of current study was absent in. We were particularly interested to see if Gram negative strict anaerobes could survive and proliferate in a biofilm grown under an oxic headspace and we were concerned that adding sucrose would cause S. mutans and other acidogenic bacteruia to dominate since P. gingivalis is not acid tolerant [30 (link)].
6
Cultivation of Anaerobic Bacteria
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A modified brain heart infusion (M‐BHI) broth was used for cultivations. BHI broth (Sigma Aldrich, USA) was supplemented with 5 mg/L hemin (Alpha Aesar, USA), 1 mg/L menadione (MP Biomedicals, LLC, France), 0.1 g/L l ‐cysteine (Sigma, USA) and 1 g/L yeast extract (Sigma, USA).
7
Oxidative Stress Modulation Assay
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Menadione (MP Biomedicals, cat. no. 102259, Solon, OH, USA) was dissolved in 100% ethanol to make 100 mM stock solution. 30% H2O2 (Avantor, cat. no. 2190-03, Radnor, PA, USA) was freshly diluted in 1× PBS to final concentrations of 150 μM or 50 μM, and then directly added to cells. α-amanitin (Cayman Chemical Co., cat. no. 17898, Ann Arbor, MI, USA) was dissolved in water to make a stock solution of 1 mg/ml and used at 1 μg/ml. 5,6-Dichlorobenzimidazole 1-β-d -ribofuranoside (DRB) (Sigma-Aldrich, cat. no. D1916) was used at 50 μM (38 (link)). The PARP inhibitor KU-0058948 hydrochloride (Axon Medchem, cat. no. 2001) and EB-47(dihydrochloride) (MedChem Express, HY-108631) were dissolved in DMSO to make a stock solution of 10 mM and used at a final concentration of 1 μM and 10 μM, respectively. α-amanitin, DRB, KU-0058948 or EB47 was added to cells 1 h before H2O2 (or Menadione) treatment, during treatment and during the 30 min recovery.
8
Transfection Reagent and Oxidative Stress Protocol
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FuGENE HD transfection reagent was purchased from Promega (Madison, WI). Menadione was purchased from MP Biomedicals (Solon, OH), and Luperox (tert-butyl hydroperoxide solution; TBOOH) was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Stock solutions of all test compounds were prepared in 100% Ethanol. MISSION LightSwitch Luciferase Assay Reagent was purchased from Sigma-Aldrich. All oligonucleotides used for gel mobility shift assays and cloning were purchased from Integrated DNA Technologies (Coralville, IA).
9
Biofilm Metabolic Viability Quantification
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Biofilm metabolic quantification for viability was done by treating with 400 μL (200 μL for LFUS) of an XTT (Biotium, Fremont, CA, USA) sodium salt/Menadione (MP Biomedical, Solon, OH, USA) solution to measure the ability of the metabolically active sessile cells to reduce the XTT to a water-soluble orange formazan compound [35 (link),58 (link)]. The plates were covered in aluminum foil and incubated in the dark for 2–3 h at 37 °C. After incubation, three 100 μL samples (one 150 μL sample for LFUS) of the resulting colored supernatant were removed and transferred into the wells of a new microtiter plate. Each plate was then read in the BioTek Synergy H1 microplate reader at 490 nm (Agilent, Santa Clara, CA, USA). The resulting absorption values were normalized to the corresponding experimental biofilm control group.
10
Oxidative stress induction in cell lines
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CS1AN and CSBWT cells were maintained in DMEM-F12 medium supplemented with 10% FBS [31 (link)]. 293T cells were maintained in DMEM supplemented with 10% FBS. All cells were maintained at 37 °C, 5% CO2. Human hTERT RPE-1 cell lines were maintained according to Hanzlikove et al. (2017) [32 (link)]. Oxidative stress was induced by treating cells with menadione (#102259, MP Biomedicals, Solon, OH, USA) in fresh medium and incubated at 37 °C for the time points indicated.
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