Cells were grown to an OD500nm of 0.6 and cytosolic cell extracts were prepared as described above. Right before starting the assay, a working solution of 25 µg/ml lactoperoxidase and 0.5 M dicarboxidine dihydrochloride (both Sigma-Aldrich) was prepared. To determine the degradation of H2O2 by cytosolic protein extracts, 5 µg of protein extract were filled up with catalase assay buffer (Catalase Assay Kit, BioVision) to 200 µl. H2O2 was added to a final concentration of 2 mM and the extracts were incubated at 30 °C. To measure the H2O2 concentration remaining in the culture at certain time points, 25 µl of the cell extract was mixed with 500 µl working solution and absorbance at 450 nm was measured as described [25 (link)]. The experiments were done in triplicates.
Catalase assay kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The Catalase Assay Kit is a laboratory tool designed to measure the activity of the enzyme catalase. Catalase is an important antioxidant enzyme found in most living organisms that catalyzes the decomposition of hydrogen peroxide into water and oxygen. This kit provides the necessary reagents and protocol to quantify catalase activity in various samples, such as cell lysates, tissues, or biological fluids.
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Lab products found in correlation
Sod assay kit, by Abcam (4 mentions)
Mda assay kit, by Abcam (2 mentions)
96 well plate reader, by BMG Labtech (2 mentions)
Lactoperoxidase, by Merck Group (1 mentions)
Mda detection kit, by Beyotime (1 mentions)
Gsh gssg assay kit, by Abnova (1 mentions)
Sod assay kit, by Solarbio (1 mentions)
Sod assay kit wst, by Dojindo Laboratories (1 mentions)
Glutathione peroxidase activity assay kit, by Abcam (1 mentions)
Alt activity assay kit, by Abcam (1 mentions)
Ammonia assay kit, by Abcam (1 mentions)
Ast activity assay kit, by Abcam (1 mentions)
Mpo activity assay kit, by Abcam (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
L glutamic acid, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Merck Group (1 mentions)
Superoxide dismutase sod assay kit, by Merck Group (1 mentions)
Human lactoferrin, by Merck Group (1 mentions)
19 protocols using catalase assay kit
1
Quantifying Catalase Activity in Cell Extracts
2
Oxidative Stress Biomarker Quantification
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MDA was detected using MDA detection kit (S0131S, Beyotime), GSH/GSSG ratio detection used GSH/GSSG Assay Kit (KA3779, Abnova), SOD detection used SOD assay Kit (BC0170, Solarbio), and catalase detection used Catalase Assay Kit (K773-100, Biovision). According to the protocol (Shen et al., 2020 (link)), cells were lysed, and reagents were added and the absorbance was measured with a microplate reader.
3
Antioxidant Enzyme Activity Quantification
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Serum was collected from whole blood by centrifugation at 3,000 g at 4℃ for 20 minutes. The levels of serum SOD activity were measured using the SOD assay kit-WST (Dojindo, Kumamoto, Japan), according to the manufacturer's instructions. A 20 µL sample solution was mixed with 200 µL of WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2.4-disulfophenyl)-2H-tetr azolium, monosodium salt) and 20 µL of xanthine oxidase in a 96-well plate, followed by incubation at 37℃ for 20 min, and the absorbance was measured at 450 nm. Levels of serum catalase activity were measured using the catalase assay kit (BioVision Inc., Milpitas, CA, USA). Catalase was first reacted with H2O2 to produce water and oxygen, the unconverted H2O2 was reacted with the OxiRedTM probe and the absorbance was measured at 570 nm. GPx activity was determined using the Glutathione peroxidase activity assay kit (BioVision Inc., Milpitas, CA, USA) according to the manufacturer's protocol, and the absorbance was measured at 340 nm.
4
Liver Enzyme and Oxidative Stress Assays
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Alanine aminotransferase (ALT), aspartate aminotransferase (AST), myeloperoxidase (MPO), and catalase (CAT) activities and the malondialdehyde (MDA) concentration were measured using an ALT activity assay kit, AST activity assay kit, MPO activity assay kit, catalase assay kit, and lipid peroxidation (MDA) assay kit (Biovision, Milpitas, CA, USA). Ammonia concentration was measured using an ammonia assay kit (Abcam, Cambridge, UK).
5
Neuronal Cell Culture and Antioxidant Assays
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Minimum essential medium (MEM), fetal bovine serum (FBS), Hank's balanced salt solution (HBSS), L-glutamic acid, L-glutamine, B-27, and neurobasal medium were purchased from Invitrogen Inc. (Grand Island, NY, USA). CORT, bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), 2-mercaptoethanol, superoxide dismutase (SOD) assay kit, formic acid, and p-coumaric acid (98% purity) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol and water were supplied by JT Baker (Deventer, Holland). The catalase assay kit was purchased from Bio-Vision Inc. (Milpitas, CA, USA). Wortmannin (681675), rapamycin (553210), H89 (371963), ZM336372 (692000), and PD98059 (513000) were purchased from Calbiochem (San Diego, CA, USA).
6
HepG2 Cell Culture and Assays
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In this study, HepG2 cells were purchased from the American Type Culture Collection (ATCC) and cultured in high glucose (4.5 mg/ml) Dulbecco's modified eagle medium (DMEM) (Sigma D6429) containing 10% fetal bovine serum (FBS) (Bio-04-127-1A), supplemented with 100 U/ml penicillin, 100 μg/ml streptomisin (Sigma P4333) in a humidified atmosphere with 5% CO 2 at 37 °C.
In this study, human lactoferrin (Sigma, USA ) , 3 -(4 , 5 -D i m e t hy l t h i a zo l-2 -y l ) -2 , 5diphenyltetrazolium bromide (MTT) assay (Biovision Catalog # K229-1000), glutathione (GSH) assay kit (Biovision Catalog # K261-100), catalase assay kit (Biovision Catalog # K773-100), lipid hydroperoxide (MDA) assay kit (Biovision Catalog # K739-100) and superoxide dismutase (SOD) assay kit (Biovision Catalog # K335-100) were used.
In this study, human lactoferrin (Sigma, USA ) , 3 -(4 , 5 -D i m e t hy l t h i a zo l-2 -y l ) -2 , 5diphenyltetrazolium bromide (MTT) assay (Biovision Catalog # K229-1000), glutathione (GSH) assay kit (Biovision Catalog # K261-100), catalase assay kit (Biovision Catalog # K773-100), lipid hydroperoxide (MDA) assay kit (Biovision Catalog # K739-100) and superoxide dismutase (SOD) assay kit (Biovision Catalog # K335-100) were used.
7
Catalase Activity in Macrophages
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Catalase activity was measured by catalase assay kit (Abcam,UK) as per manufacturer’s instructions. Briefly, PMA differentiated macrophages were treated with OxLDL in the presence and absence of cyclophilin A (100 ng/mL) for 24 h. Cells were harvested after treatment, washed with cold PBS twice and homogenized with 200 µL of cold assay buffer. To start the reaction 12 µL of 1 mM H2O2 was added and incubated for 30 min at 25 °C. The unconverted H2O2 was measured calorimetrically using OxiRed probe at 530 nm using microplate reader (Biorad laboratories, Germany). Catalase activity was calculated as the amount of H2O2 decomposed per minute at pH 4.5 at 25 °C.
8
Catalase Activity Quantification
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Catalase activity was determined by the Catalase Assay kit (Abcam Ref. ab83464). In the assay H2O2 was added to the catalase samples in order to measure the unconverted H2O2 which reacts with OxiRed™. Reaction final product was measured at Ex/Em = 535/587 nm by Victor 3 Multilabel Plates Reader. Catalase activity is reversely proportional to the signal.
9
Oxidative Stress Biomarkers Quantification
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Plasmatic lipid peroxidation as a consequence of oxidative stress was estimated by measuring thiobarbituric acid reactive substances (TBARS) using a kit (OxiSelect™ TBARS Assay Kit-MDA Quantitation, Cell Biolabs Inc., San Diego, CA, USA) according to the manufacturer’s instructions, and expressed in µmol/L malondialdehyde (MDA). Superoxide dismutase (SOD) and catalase activities were measured according to the manufacturer’s instructions (Superoxide dismutase assay kit and Catalase Assay Kit, Abcam, Paris, France) and expressed, respectively, in percent of inhibition rate and μmol/L. Total antioxidant capacity (TAOC) with the radical cation ABTS•+ (2,2′-azino-bis-(3)-ethylbenzthioazoline-6-sulfonic acid, VWR, Fontenay sous Bois, France) was determined using 6-hydroxy-2, 5, 7, 8-tetramethylchromane-2-carboxylic acid (Trolox; Sigma-Aldrich, St Quentin Fallavier, France) equivalent, as previously described for plasma [1 (link)].
10
Catalase Activity Assay for ω-3 Fatty Acids
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To measure the activity of anti-oxidant enzyme catalase, H19-7 cells treated with different ω-3 fatty acids and induced with Aβ1-40 were collected. After washing with PBS, the catalase enzyme activity of samples was analyzed spectrophotometrically at 570 nm by using a Catalase Assay Kit (Abcam, Cambridge, MA, USA) according to manufacturer’s instructions. Briefly, catalase first reacts with H2O2 to produce water and oxygen. Thereafter, the unconverted H2O2 will react with OxiRed probe to formulate a product which can be measured at 570 nm. A standard concentration of hydrogen peroxide was used to create a calibration curve and the results of the assay were expressed as microunits per microgram protein.
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