The serum and hepatic contents of lipid peroxidation product were measured using a Thiobarbituric Acid Reactive Substances (TBARS) assay kit (ZeptoMetrix, Buffalo, NY, USA). The levels of the lipid peroxidation products were presented as Malondialdehyde (MDA) equivalents.
Tbars assay kit
Manufactured by ZeptoMetrix
Sourced in United States
The TBARS Assay Kit is a laboratory test used to measure the levels of thiobarbituric acid reactive substances (TBARS) in biological samples. TBARS are a biomarker for oxidative stress and serve as an indicator of lipid peroxidation. The kit provides a colorimetric assay to quantify TBARS concentrations.
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Lab products found in correlation
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Protease inhibitor cocktail, by Merck Group (1 mentions)
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H2o2 assay kit, by Abcam (1 mentions)
Hand homogenizer, by As One (1 mentions)
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Mutanolysin, by Merck Group (1 mentions)
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16 protocols using tbars assay kit
1
Lipid Peroxidation Quantification
2
Lipid Peroxidation Measurement Protocol
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The prefrontal cortical tissues, serum, and urine were isolated and subjected to measurement of lipid peroxidation products using a Thiobarbituric Acid Reactive Substances (TBARS) assay kit (ZeptoMetrix, Buffalo, NY, USA). The calculated levels of lipid peroxidation products were expressed as MDA equivalents, according to the manufacturer’s instructions.
3
Oxidative Stress Biomarkers in Plasma and Tissue
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To detect malondialdehyde (MDA), lipid peroxidation in plasma was measured with a TBARS Assay Kit (ZeptoMetrix, Buffalo, NY, USA), according to the manufacturer’s instructions. Plasma SOD activity was measured with a SOD Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s protocol.
Colon rectum tissues were collected and homogenized in 500 μL cold reaction buffer supplemented with a protease inhibitor cocktail (Sigma Aldrich). After centrifugation (4°C, 15 minutes, 16,000 ×g), supernatants were stored at −80°C. Catalase activity was detected by using an Amplex Red® Catalase Assay Kit (Invitrogen, Carlsbad, CA, USA.) according to the manufacturer’s instructions.
Colon rectum tissues were collected and homogenized in 500 μL cold reaction buffer supplemented with a protease inhibitor cocktail (Sigma Aldrich). After centrifugation (4°C, 15 minutes, 16,000 ×g), supernatants were stored at −80°C. Catalase activity was detected by using an Amplex Red® Catalase Assay Kit (Invitrogen, Carlsbad, CA, USA.) according to the manufacturer’s instructions.
4
Lipid Peroxidation Quantification Protocol
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A thiobarbituric acid reactive substance (TBARS) assay kit (ZeptoMetrix Corporation, Buffalo, NY, USA) was used to measure the lipid peroxidation products, MDA equivalents. The formation of lipid peroxides was measured in the homogenates of the hepatic tissues. The formation of MDA, an end product of fatty acid peroxidation, was measured spectrophotometrically at 532 nm using a TBARS assay, and levels of MDA were expressed as nmol/mg tissue.
5
Biomarker Quantification in Mice
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Immediately after killing the mice, blood was collected via cardiac puncture and was centrifuged forthwith under refrigeration to obtain plasma for the measurement of insulin, malondialdehyde (MDA), TNF-a, adiponectin, monocyte chemotactic protein-1 and C-reactive protein. Insulin and adiponectin levels were assessed using the Mouse Insulin and the Adiponectin ELISA Kits, respectively (Linco Research). MDA was assessed using the TBARS Assay Kit (ZeptoMetrix Corporation). The level of TNF-a was measured using the specific Mouse TiterZyme ELISA Kits from Assay Designs, Inc.. Plasma C-reactive protein levels were measured using the Mouse C-reactive protein ELISA Kit (Life Diagnostics, Inc.).
6
LDL Isolation and Oxidation for Endothelial Cell Assays
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LDL was isolated from human plasma obtained from a local blood bank—Lifesource (now Vitalant, Chicago, IL), as described in our previous studies (7 (link)). Briefly, LDL was isolated by sequential centrifugation in potassium bromide (KBr, Acros Organics; Thermo Fisher Scientific, Waltham, MA), and dialyzed to remove KBr and EDTA. Oxidized LDL (oxLDL) was prepared by Cu2+ as previously described (8 (link)). The degree of oxidation was assessed by the TBARS (thiobarbituric acid reactive substances) content of LDL and was measured using a TBARS Assay Kit (ZeptoMetrix, Buffalo, NY) as expressed with malondialdehyde (MDA) equivalent. Experiments were performed with Human Aortic Endothelial cells (HAECs; Lonza, Allendale, NJ), grown in standard conditions. Serum-starved cells were treated with either 10 μg/ml oxLDL, 50 mg/dl LDL, or 250 mg/dl LDL for 24 h, then lysed in methanol and stored at −80°C.
7
Serum ALT, Cytokines, and Oxidative Stress
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Serum alanine aminotransferase (ALT) activity was determined using a kinetic method (TECO Diagnostics, Anaheim, CA). Intracellular cytokine levels were monitored in liver whole cell lystate using TNFα, IL-6, MCP-1 ELISA kits (Biolegend, San Diego, CA). Liver thiobarbituric acid reactive substances (TBARS), a measure of oxidative stress, were estimated using TBARS assay kit (ZeptoMetrix, Buffalo, NY). Tissue IL-17 and serum AFP were assayed by ELISA (R&D systems, Minneapolis, MN).
8
Lipid Peroxidation Quantification
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The prefrontal cortical tissues, serum, and 24 h urine samples were collected and isolated. The levels of lipid peroxidation products were measured using a Thiobarbituric Acid Reactive Substances (TBARS) assay kit (ZeptoMetrix, Buffalo, NY, USA). The calculated levels of the lipid peroxidation products were expressed as MDA equivalents according to the manufacturer’s instructions.
9
Hepatic Oxidative Stress Biomarkers
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Commercial assay kits for the examination of hepatic malondialdehyde (MDA; #A003-1-2), catalase (CAT; #A007-1-1), superoxide dismutase (SOD; #A001-3-2) and glutathione (GSH; #A006-2-1) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Hydrogen peroxide (H2O2) levels in liver were tested using commercially available kit (#S0038; Beyotime Biotechnology) in line with the provider suggested. Hepatic lipid peroxidation was examined by thiobarbituric acid reactive substances (TBARS) formation [32 (link)]. TBARS Assay Kit (#801192, ZeptoMetrix Corporation, USA) was used for the measurements of liver TBARS contents following the manufacturer's protocols.
10
Plasma Biomarkers Measurement
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The plasma levels of NO, IL-6, and TNF-alpha were determined as described above. The plasma MDA concentration was measured using the TBARS Assay Kit (ZeptoMetrix Corp., New York, USA) according to the manufacturer's instructions.
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