Tbars assay kit

Manufactured by Cayman Chemical

Sourced in United States, United Kingdom, Estonia, Japan

The TBARS assay kit is a laboratory tool used to measure the levels of thiobarbituric acid reactive substances (TBARS) in biological samples. TBARS are commonly used as a biomarker for oxidative stress and lipid peroxidation. The kit provides the necessary reagents and protocols to perform this analysis.

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394 protocols using tbars assay kit

Lipid Peroxidation and Antioxidant Enzyme Evaluation

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MDA levels in tissues were evaluated using commercially available assay kits to determine the lipid peroxidation state (TBARS Assay Kit, item no. 10009055, Cayman Chemicals, Ann Arbor, MI, USA). The measuring principle is based on the reaction with thiobarbituric acid (TBA) in boiling water for 60 min in an acidic medium and the measurement of the absorbance of the reaction mixture at 532 nm (Ohkawa et al., 1979). A VersaMax Tunable Microplate Reader was used to measure the absorbance (Molecular Devices, San Jose, CA, USA). MDA concentrations in tissues were expressed as nmol MDA/mg protein.
In tissues, GPx and SOD activity were evaluated using ready-to-use assay kits. (RANSEL RS505, RANSOD SD125, Randox Laboratories Ltd., Crumlin, UK) by using an automated BS-240 VET Clinical Chemistry Analyser (Mindray, Shenzhen, China). The quantities of GPx and SOD in the tissue samples (stomach, small intestine, and large intestine) were expressed as U/mg protein.

Ceylanlı D., Şehirli A.Ö., Gençosman S., Teralı K., Şah H., Gülmez N, & Sayıner S. (2022). Protective Effects of Alpha-Lipoic Acid against 5-Fluorouracil-Induced Gastrointestinal Mucositis in Rats. Antioxidants, 11(10), 1930.

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Quantifying Lipid Peroxidation in Brain Tissues

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Lipid peroxidation, in brain tissues, was determined using TBARS assay kit (Cayman Chemical, MI) according to the manufacturer’s instructions. Briefly, MDA standard curve was prepared by diluting 250 μL MDA standard with 750 μL water and then serial dilution that started from 0 μm to 50 μm was prepared. A mixture of 100 μL of the serum sample, 100 μL of homogenate brain tissues in cold 10 mM Tris-HCl (pH 7.5), standard and 100 μL of SDS was first prepared. Four milliliters of color reagent was added to each mixture and boiled for an hour. After that, the reaction was stopped on ice for 10 min and centrifuged for 10 min at 1600×g; then 150 μL of the supernatant was loaded in a 96-well plate and absorbance was read at 540 nm. TBARS concentration was calculated from MDA standard curve.

Almutairi M.M., Alanazi W.A., Alshammari M.A., Alotaibi M.R., Alhoshani A.R., Al-Rejaie S.S., Hafez M.M, & Al-Shabanah O.A. (2017). Neuro-protective effect of rutin against Cisplatin-induced neurotoxic rat model. BMC Complementary and Alternative Medicine, 17, 472.

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Quantifying Lipid Peroxidation via TBARS

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Malondialdehyde, a product of lipid peroxidation, was quantified by the thiobarbituric acid reactive substances (TBARS) Assay Kit (Cayman Chemical) as described (Yamane et al., 2014 (link)). In brief, cells scraped into PBS containing complete protease inhibitor cocktail (Roche) were homogenized by sonication on ice using BioRuptor (Diagenode). The amount of malondialdehyde in 100 μl of cell homogenates was analyzed by a fluorescent method as described by the manufacturer. Lipid peroxidation levels were expressed as the amount of malondialdehyde normalized to the amount of total protein.

Yamane D., Hayashi Y., Matsumoto M., Nakanishi H., Imagawa H., Kohara M., Lemon S.M, & Ichi I. (2021). FADS2-dependent fatty acid desaturation dictates cellular sensitivity to ferroptosis and permissiveness for hepatitis C virus replication. Cell chemical biology, 29(5), 799-810.e4.

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Serum Lipid Peroxidation Assessment

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Lipid peroxidation and oxidative stress were assessed from serum samples using a TBARS assay kit (Cayman, Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions.

Yuan Y., Naito H., Kitamori K., Hashimoto S., Asano T, & Nakajima T. (2020). The antihypertensive agent hydralazine reduced extracellular matrix synthesis and liver fibrosis in nonalcoholic steatohepatitis exacerbated by hypertension. PLoS ONE, 15(12), e0243846.

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Renal Tissue MDA Quantification

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The MDA concentration in renal tissues was measured according to a previously published protocol by our group (32 (link)). In short, homogenization and centrifugation of the renal tissues were performed at 8,832 x g for 10 min at 4˚C and the supernatant was collected and stored at -80˚C for MDA analysis. Subsequently, the MDA content of the renal tissues was measured according to the instructions of a commercial kit (TBARS assay kit; Cayman Chemical).

Jawad A., Yoo Y.J., Cho J.H., Yoon J.C., Tian W., Islam M.S., Lee E.Y., Shin H.Y., Kim S.E., Kim K., Ahn D., Park B.Y., Kim I.S., Lee J.H, & Tae H.J. (2021). Therapeutic hypothermia effect on asphyxial cardiac arrest-induced renal ischemia/reperfusion injury via change of Nrf2/HO-1 levels. Experimental and Therapeutic Medicine, 22(3), 1031.

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Oxidative Stress Biomarkers in Plasma

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Malondialdehyde (MDA) (TBARS Assay Kit, Cayman, MI, USA; cat#10009055), glutathione (GSH) (Glutathione Assay Kit, Cayman, cat#703002), superoxide dismutase (SOD) (Superoxide Dismutase Assay Kit, Cayman, cat#706002), free fatty acids (FFA) (Free Fatty Acid Fluorometric Assay Kit, Cayman, cat#700310), and ferrous iron (Fe²⁺) (Iron Colorimetric Assay Kit, Biovison, CA, USA; cat# K390) in plasma were measured by ELISA following the manual instructions. Before measurement of GSH, the plasma should be deproteinised as instructed in the manufacturer's protocol.

Xiao Q., Yan L., Han J., Yang S., Tang Y., Li Q., Lao X., Chen Z., Xiao J., Zhao H., Yu F, & Zhang F. (2022). Metabolism-dependent ferroptosis promotes mitochondrial dysfunction and inflammation in CD4+ T lymphocytes in HIV-infected immune non-responders. eBioMedicine, 86, 104382.

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Oxidation and Copper Removal of HDL

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Native HDL was obtained commercially from Sigma-Aldrich Inc. (St. Louis, MO, USA) and oxidized as previously described [24] with modifications. Briefly, HDL at a final concentration of 0.5 mg/ml was incubated at 37 °C for 16 h in the presence of 50 μM CuSO 4 in PBS. The reaction was stopped by storing the oxHDL at 4 °C to prevent further oxidation. The extent of lipoprotein oxidation was monitored by measuring the thiobarbituric acid reactive substances (TBARS) formation [28] with the TBARS assay kit (Cayman Chemical Company, Ann Arbor, MI USA) as recommended by the supplier. For oxHDL, a total of 12.13 ± 1.04 μM MDA was obtained vs. 2.46 ± 0.65 μM from Native HDL, in a total of seven independent experiments (P ≤ 0.001). To chelate copper from the reaction, oxHDL was incubated for 5 min with 100 mg/ml CHELEX-100 (Bio-Rad Laboratories Inc., Hercules, CA USA), centrifuged at 4 °C for 1 min at 500 × g, and the pellet was discarded [29] .

Pérez L., Vallejos A., Echeverria C., Varela D., Cabello-Verrugio C, & Simon F. (2019). OxHDL controls LOX-1 expression and plasma membrane localization through a mechanism dependent on NOX/ROS/NF-κB pathway on endothelial cells. Laboratory investigation; a journal of technical methods and pathology, 99(3).

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Antioxidant Enzyme and Lipid Peroxidation Assay

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Catalase (CAT) activity was studied by measuring breakdown of H₂O₂ at 240 nm [12 ], and peroxidase (PX) activity was measuring the oxidation of guiacol at 436 nm according to standardized methods [13 (link)]. The extent of lipid peroxidation was measured in terms of serum malondialdehyde (MDA) content by colorimetric thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical, USA) as per the manufacturer's instructions.

Dey P., Saha M.R., Roy Choudhuri S., Sarkar I., Halder B., Poddar-Sarkar M., Sen A, & Chaudhuri T.K. (2019). Oleander Stem and Root Standardized Extracts Mitigate Acute Hyperglycaemia by Limiting Systemic Oxidative Stress Response in Diabetic Mice. Advances in Pharmacological Sciences, 2019, 7865359.

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Serum Markers of Liver Injury

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Blood samples were obtained from the right heart ventricle. Serum ALT level was determined to assess the liver injury by using commercial kits (Bio Vision, CA) according to the manufacturer’s instructions. Serum MDA formation assay was performed using the TBARS assay kit (Cayman, USA) according to the manufacturer’s instructions.

Masuda Y., Vaziri N.D., Takasu C., Li S., Robles L., Pham C., Le A., Vo K., Farzaneh S.H., Stamos M.J, & Ichii H. (2014). Salutary effect of pre-treatment with an Nrf2 inducer on ischemia reperfusion injury in the rat liver. Gastroenterology and hepatology, 1(1), 1-7.

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Quantifying Lipid Peroxidation via TBARS

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Thiobarbituric acid reactive substances assay (TBARS) was performed to analyze lipid peroxidation as per manufacturer’s instructions (Cayman Chemical TBARS Assay Kit). For this assay, PC3 cells were seeded in T25 flasks with the seeding density of 5 × 10⁵ and treated with varying concentrations of NCX4040 in the presence or absence of catalase and DETDC for 6 hrs. After treatment, adherent and suspension cells were collected and centrifuged 1000g for 5min. Following centrifugation, supernatant was discarded and pellet was suspended in 1ml of culture medium and cells were subjected to sonication on ice. 100 μl of sample were mixed with equal volume of SDS and 4 ml of thiobarbituric acid reagent and the mixture was heated for 1 hr at 75°C. After 1 hr, the mixture was incubated on ice for 10min. After incubation, samples were centrifuged for 10 min at 1600 × g at 4°C. Fluorescence of the processed samples were read at an excitation wavelength of 530 nm and an emission wavelength of 550 nm in spectrophotometer (Biotech Synergy 2). Reaction mixture was used as a blank control. A standard curve was plotted with tetramethoxypropane (which is equivalent to the same concentrations of MDA) with concentrations ranging from 0.0625 to 50 (μM. Results of lipid peroxidation assay presented as malondialdehyde (MDA) concentration ((μM) per sample

Chinnapaka S., Zheng G., Chen A, & Munirathinam G. (2019). Nitro aspirin (NCX4040) induces apoptosis in PC3 metastatic prostate cancer cells via hydrogen peroxide (H2O2)-mediated oxidative stress. Free radical biology & medicine, 143, 494-509.

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