This endogenous antioxidant enzyme detoxifies the free radical hydrogen peroxide, which is toxic to cells. The Cayman Chemical catalase assay (catalogue no. 707002) method works based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2O2. The formaldehyde produced during the above reaction is measured colorimetrically using chromagen purpald. The steps to perform the assay were followed on the five sample groups of the CCl4-induced liver injury supernatant homogenate as per the manual in the kit. The absorbance was read at 540 nm using a plate reader.
Catalase assay
Manufactured by Cayman Chemical
Sourced in United States
The catalase assay is a laboratory tool used to measure the activity of the enzyme catalase. Catalase is an important antioxidant enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen. The assay provides a quantitative assessment of catalase function.
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4 protocols using catalase assay
1
Catalase Activity Assay for Oxidative Stress
2
Catalase Activity Assay in Caveolin-1 Knockdown Cells
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Cells grown in 6-well dishes were transfected with control or caveolin-1 siRNA. Forty eight hours after transfection, media was replaced by fresh cell culture media containing either 10 mM 3-amino-1,2,4-triazole (ATZ) or vehicle, and incubated for additional 21 hrs. Cells were then rinsed with PBS, scraped into 0.5 ml of potassium phosphate buffer (50 mM, pH 7) containing 1 mM EDTA and protease inhibitors cocktail. Cells were sonicated and centrifuged (13,200 rpm×15 min), and catalase activity was assayed in the supernatants using a catalase assay (Cayman Chemicals) according to the manufacturer’s instructions.
3
Catalase Activity Measurement in Plasma
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CAT activity was evaluated in the plasma of peripheral blood. Twenty-five µL samples were centrifuged at 1000× g for 10 min at 4 °C to obtain plasma. The activity of CAT was evaluated according to the instructions for the Cayman Chemicals Catalase Assay (No. 707002; Ann Arbor, MI, USA). This kit uses the peroxidation function of CAT to determine enzyme activity. The absorbance was read at 540 nm in a Multiskan™ FC microplate reader (Thermo Scientific™, Vantaa, Finland). One unit of CAT was defined as the amount of enzyme that induced the formation of 1 nmol of formaldehyde/min. CAT activity was measured in duplicate (per sample) and according to the CAT standard curve.
4
In Vivo and In Vitro Evaluation of (R)-(−)-carvone
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(R)-(−)-carvone and tyloxapol (Triton WR 1339) were purchased from Sigma-Aldrich (USA) and fenofibrate (Trigless) was purchased from Jordan Sweden Medical and Sterilization Company (JOSWE), Jordan. Kits for glucose (code: 11503), plasma TGs (code: 11529), total cholesterol (TC) (code: 11505), high-density lipoprotein cholesterol (HDL-C) (code: 11648) and low-density lipoprotein cholesterol (LDL-C), and precipitating reagent (code: 11579) were purchased from BioSystems (S.A., Barcelona Spain). Total glutathione (GSH) (catalog number: 703002) and catalase assay (catalog number: 707002) were supplied by Cayman Chemical Co., USA. The insulin-secreting human pancreatic β-cell line 1.1E7 was obtained from the European Collection of Cell Cultures (ECACC), United Kingdom. The MTT kit was from Promega (USA). All other chemicals were of analytical grade. (R)-(−)-carvone was emulsified in 2% Tween 20 for in vivo studies. For in vitro studies, (R)-(−)-carvone was dissolved in dimethyl sulfoxide (DMSO) and diluted to prepare the various concentrations so that the final DMSO concentration was less than 0.5%. Fenofibrate and tyloxapol were dissolved in normal saline. All reagents were freshly prepared before use.
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