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15 protocols using catalase cat

1

Assessing Antioxidant Responses in Plants

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The samples of B. napus and L. cruciata were homogenized with Phosphate Buffered Saline (PBS) solution (Sigma-Aldrich) (0.1 mL of 50 mM, pH 7.4) using a sterile pestle. The protein extract was used to evaluate the levels of ROS and the activity of the antioxidant enzymes.
ROS levels were assessed using 2′,7′-dichlorofluorescin diacetate (H2DCFDA). The extract was incubated with 5 μM H2DCFDA for 30 min at 37 ± 1 °C. ROS quantity was monitored by fluorescence (excitation wavelength of 350 nm and an emission wavelength of 600 nm).
The activity of the antioxidant enzymes catalase (CAT) (Sigma-Aldrich Co., St Louis, MO, USA), Superoxide dismutases (SOD) (19160, Sigma, St Louis, MO, USA), and glutathione S-transferases (GST) (CS0410, Sigma St Louis, MO, USA) were measured following the kit instructions. The level of ROS and the antioxidant activity enzyme was detected using a microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) [35 (link)]. For each sample, 3 replicates were performed.
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2

Perfluorinated Compound Synthesis and Characterization

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IR780 iodide (purity ≥ 95.0%) was purchased from Bellingway Technology Co., Ltd. (Beijing, China). Bardoxolone methyl (CDDO-Me) and perfluoropolyether with molecular weight 6000 (PFPE MW 6000) were purchased from Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Catalase (CAT, 2000–5000 units/mg protein) from bovine liver was obtained from Sigma-Aldrich® (Shanghai, China). Minimum essential medium (MEM), Ham’s F-12K medium, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and Tween 20 and acetonitrile (purity ≥ 99.9%) were purchased from Chaoyuan Zhicheng Biotechnologies Co., Ltd. (Guiyang, China). Fomblin® Y (MW 1800), 1H-tridecafluorohexane (MW 320.05), perfluorooctane (MW 438.06), and 1-bromoheptadecafluorooctane (MW 498.96) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Syringe filters with a pore size of 0.22 μm and 0.45 μm (PTFE hydrophilic) were purchased from Navigator Lab Instrument Co., Ltd. (Tianjin, China). Deionized water was produced by ELGA VEOLIA (Veolia Water Solutions & Technologies, Shanghai, China).
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3

Genotoxicity Studies with TK6 Cells

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Lymphocytes are the cell type most often used when investigating DNA damage [36 (link)]. Especially the human TK6 lymphocyte cell line has been widely utilized in genotoxicity studies [37 (link), 38 (link)]. For this reason, we used TK6 (ATCC CRL-8015) cells, a p53-competent, human lymphoblast cell line. Cells were cultured in Roswell Park Memorial Medium without phenol red (RPMI1640; PanBioTech) supplemented with 10% fetal bovine serum, 2% glutamine, and 1% penicillin/streptomycin (all Sigma). All incubations were performed in cell culture conditions (CB210; Binder) at 37°C, 95% humidity, and 5% carbon dioxide. As ROS scavengers, catalase (cat; 20 μg/ml), glutathione (GSH; 1 mM), or superoxide dismutase (SOD; 100 U/ml) was used (all Sigma). As enzyme or signaling inhibitors, Z-VAD-FMK (R&D Biosciences), SB202190 (Sigma), KU55933 (SelleckChem), Ly294002 (Cell Signaling Technologies), wortmannin (InvivoGen), or SP600125 (Santa Cruz Biotechnology) was used at different concentrations and incubated with cells for 1 h prior ROS or UV treatment. Final concentrations for a selected choice of inhibitors were 1 μM for KU55933, 1 μM for SB202190, and 25 μM for Z-VAD-FMK.
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4

Antioxidant Activity Determination

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Gallic acid, Folin-Ciocalteu reagent, catechin, rutin, thiobarbituric acid, catalase (CAT), and malondialdehyde (MDA) were purchased from Sigma–Aldrich Co. (Sigma, St. Louis, USA). Standards (iron (Fe), zinc (Zn), calcium (Ca), potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), and sodium (Na)) were obtained from Merck (Darmstadt, Germany). Superoxide dismutase (SOD) and glutathione peroxidase (GPx) assay kits were purchased from Randox Laboratory Ltd. Methanol was purchased from PanReac (Barcelona, Spain). All other chemicals and solvents were of analytical grade and were obtained from Sigma–Aldrich (Sigma, St. Louis, USA).
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5

Uranyl Acetate and Arsenic Exposure

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Uranium, as uranyl acetate (UA) (99.6% purity) (Electron Microscopy Science, PA, USA) was comprised of 99.9% 238U and 0.1% 235U according to the product’s technical bulletin. Uranyl acetate was chosen because the uranyl ion has a +VI valence in aqueous medium, mimicking an environmental exposure. UA had a radioactive activity of 0.51 μCi g−1 and was handled according to the regulations set forth by the Radiation Safety office at the University of New Mexico. Arsenic in the form of sodium arsenite (AS) (99% purity) (Fluka Chemie, Buchs, Germany) was used for experimental treatments. Ten millimolar stock solutions of UA and one hundred millimolar stock solutions of AS were prepared in MilliQ water and sterilized using a 0.22-μm syringe filter. Working solutions were prepared by diluting the stock with complete cell growth medium. Etoposide (ETOP) (Millipore Sigma, MA, USA) was suspended in dimethylsulfoxide (DMSO) (Sigma-Aldrich St. Louis, MO) at a concentration of 100 mM and stored at −20°C protected from light. Catalase (Cat) (Sigma-Aldrich, MO, USA), lyophilized from bovine liver, was made fresh before use by combining 0.5 mg of Cat with 5 mL of RPMI medium. The solution was filter sterilized using 0.2 μm membrane filter.
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6

Multiparametric Evaluation of Cell Death

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Caspase activity was evaluated using the luminescent kit Caspase-Glo™3/7 Assay (Promega Corporation, Fitchburg, Wisconsin, USA). Morphological membrane changes were detected using Annexin V-EGFP/PI detection kit (Biovision, Mt. View, CA, USA). Viability was measured using the colorimetric CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega). The CellTiter 96® Aqueous One Solution Reagent contains the tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and an electron coupling reagent (1-methoxy phenazine methosulfate—PMS). The liquid scintillation cocktail was the Ready-Gel (Beckman Instrument, Fullerton, CA, USA). The proteasome inhibitors PS-341 and MG-132, the necroptosis inhibitor Necrostatin-1 and the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]fluoromethylketone (Z-VAD-fmk) were supplied by Vinci-Biochem (Florence, Italy). The hydrogen peroxide scavenger catalase (CAT) and fludarabine (FLU) were purchased by Sigma-Aldrich. For SDS-PAGE, precasted gels and buffer strips obtained from GE Healthcare were used. Other reagents used were from Merck (Darmstadt, Germany), Carlo Erba (Milano, Italy) and Sigma-Aldrich.
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7

Intracellular ROS Measurement Protocol

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Intracellular ROS was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA, Eugene, OR, United States). Briefly, L929 cells were treated with 100 nM CNP for 24 h or 100 μM NAC, 100 UI SOD (Sigma-Aldrich, St. Louis, MO, United States) and 100 UI catalase (CAT, Sigma-Aldrich, St. Louis, MO, United States) for 1 h and irradiated with UVA at a unique dose of 30 J/cm2 or with 15 J/cm2 followed by an additional dose (15 J/cm2) after 24 h. After different times, cells were incubated with 5 μM H2DCF-DA for 30 min in the dark at 37°C. Cell-associated fluorescence was detected using a spectrofluorimeter (VICTOR X3, PerkinElmer, United States, λex = 488 nm, λem = 525 nm). The fluorescence percentage was expressed as arbitrary fluorescence units per μg of protein determined by Bradford method (Bio-Rad, CA, United States).
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8

Preparation and Characterization of Palladium Nanoparticles

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PdCl2 for the preparation of the PdNPs was purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin-streptomycin solution, trypsin-ethylenediaminetetraacetic acid (EDTA) solution, Dulbecco’s modified Eagle’s medium (DMEM, F-12), and 1% antibiotic-antimycotic solution were obtained from Life Technologies (Carlsbad, CA, USA). A fetal bovine serum (FBS) in vitro toxicology assay kit was purchased from Sigma-Aldrich. The reagent kits for the measurement of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) were purchased from Sigma-Aldrich.
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9

Spin Traps for Radical Detection

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Four spin traps were selected to cover a large range of lipophilicity and reactivity against the main families of radicals. DEPMPO and EMPO were obtained from Radical Vision (Marseille, France). POBN and PBN were purchased from Enzo-Alexis Biochemicals (Lausanne, Switzerland). Spin traps were dissolved in water, except for PBN which was dissolved in DMSO. Stock solutions (2 M) were stored at -80°C.
Chemicals for the radical generating systems (xanthine, xanthine oxidase, diethylenetriaminepentaacetic acid (DTPA), ferrous ammonium sulfate (Fe (NH4)2(SO4)2), hydrogen peroxide (H2O2), sodium sulfite, sodium dichromate, sodium azide and horseradish peroxidase) were purchased from Sigma Aldrich (St Louis, MO).
Toxic agents were chosen in order to produce radicals representative of the main types commonly observed (O, N, C or S-centered radicals). Menadione bisulfite, hydrogen peroxide, tert-butylhydroperoxide, phenylhydrazine, catalase (CAT) and superoxide dismutase-polyethylene glycol (PEG-SOD) were obtained from Sigma Aldrich (St Louis, MO).
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10

Evaluating Cell Viability during Chlamydia Infection

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The cell viability after CT infection was also evaluated on cells pre-treated with 100 μM of the pan-caspase inhibitor Z-Vad-fmk (Z-VAD), 100 μM of the necroptosis inhibitor necrostatin-1 (Nec) and 100 U/ml of the hydrogen peroxide scavenger catalase (CAT) (Sigma Aldrich, Missouri, USA).
To evaluate the involvement of caspase-1 during chlamydial infection, the cell viability was also evaluated on cells pre-treated with 100 μM of the selective caspase-1 inhibitor Ac-YVAD-cmk (Sigma Aldrich).
Cells (1 × 104/well) were seeded in a 96-well plate in 100 μL of complete medium and allowed to reach the 60% of confluence. Inhibitors or scavenger were added 3 h before the infection with CT at MOI 3 and plates were centrifuged at 640 × g for 2 h and incubated at 37 °C with 5% CO2. After a 72 h-incubation, cell viability was evaluated as above described.
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