Oxidative stress was determined by measuring myocardial levels of lipid and protein peroxidation. Lipid peroxidation was determined by measuring myocardial levels of thiobarbituric acid reactive substances (TBARS) using a competitive enzyme immunoassay with a plate reader (Thermo Scientific Multiskan Spectrum, Rockford, IL, USA). Measurements were obtained using a commercially available TBARS kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s instructions. Protein carbonyl content was determined by the reaction between 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls, forming a Schiff base with a plate reader. Measurements were obtained using a commercially available protein carbonyl assay kit (Cayman Chemical), according to the manufacturer’s instructions. The absorbance of the standard and samples was read with a plate reader. All measurements were performed in duplicate on the same microtiter plate with the same setting. The amount of TBARS was normalised to the total amount of myocardium in the sample and calculated in picogram∙mg-1 protein. The protein carbonyl content was calculated in nmol∙mg-1 protein.
Tbars kit
Manufactured by Cayman Chemical
Sourced in United States
The TBARS (Thiobarbituric Acid Reactive Substances) kit is a colorimetric assay used for the quantitative determination of lipid peroxidation in biological samples. The kit measures the presence of malondialdehyde, a byproduct of lipid peroxidation, by its reaction with thiobarbituric acid.
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Lab products found in correlation
Multiskan spectrum, by Thermo Fisher Scientific (1 mentions)
Protein carbonyl assay kit, by Cayman Chemical (1 mentions)
Pge2 monoclonal enzyme immunoassay kit, by Cayman Chemical (1 mentions)
Griess reagent kit, by Thermo Fisher Scientific (1 mentions)
Sod assay kit, by Cayman Chemical (1 mentions)
Oxiselect oxidative dna damage elisa kit, by Cell Biolabs (1 mentions)
Ab42789, by Abcam (1 mentions)
Antioxidant assay kit, by Cayman Chemical (1 mentions)
2 7 dichlorofluorescein diacetate dcf da, by Thermo Fisher Scientific (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Rosmarinic acid, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Griess assay kit, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
20 protocols using tbars kit
1
Myocardial Oxidative Stress Biomarkers
2
Lipid Peroxidation Assay in Mouse Tissues
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The mouse tissues were sonicated
in RIPA buffer with protease inhibitor for 3 cycles of 15 s each on
ice. The tubes were centrifuged at 14 000 rpm for 10 min at
4 °C. The supernatant was used for the lipid peroxidation assay.
The assay was performed using the TBARS kit (Cayman Chemicals, U.S.A.)
as per the manufacturer’s instructions. Briefly, samples or
standard MDA was mixed with 4 mL of assay reagent (containing TBA
acetic acid and TBA sodium hydroxide). The vials were tightly capped
and kept in a boiling water bath for 1 h and subsequently transferred
to ice for 10 min to quench the reaction. The vials were centrifuged
at 14 000 rpm for 10 min at 4 °C. From each vial, 150
μL of the supernatant was transferred to a clear 96-well plate,
and the absorbance was read at 530 nm and corrected using a blank.
The corrected absorbance of MDA was plotted as a function of MDA concentration,
and the concentration of MDA for each sample was extrapolated from
the standard curve. MDA (μM) = [corrected absorbance –
y intercept/slope]. ANOVA (analysis of variance) was performed to
test the significance of change in different biological groups at
different time points.
in RIPA buffer with protease inhibitor for 3 cycles of 15 s each on
ice. The tubes were centrifuged at 14 000 rpm for 10 min at
4 °C. The supernatant was used for the lipid peroxidation assay.
The assay was performed using the TBARS kit (Cayman Chemicals, U.S.A.)
as per the manufacturer’s instructions. Briefly, samples or
standard MDA was mixed with 4 mL of assay reagent (containing TBA
acetic acid and TBA sodium hydroxide). The vials were tightly capped
and kept in a boiling water bath for 1 h and subsequently transferred
to ice for 10 min to quench the reaction. The vials were centrifuged
at 14 000 rpm for 10 min at 4 °C. From each vial, 150
μL of the supernatant was transferred to a clear 96-well plate,
and the absorbance was read at 530 nm and corrected using a blank.
The corrected absorbance of MDA was plotted as a function of MDA concentration,
and the concentration of MDA for each sample was extrapolated from
the standard curve. MDA (μM) = [corrected absorbance –
y intercept/slope]. ANOVA (analysis of variance) was performed to
test the significance of change in different biological groups at
different time points.
3
Quantification of Serum and Tissue MDA Levels
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The serum and tissue MDA concentrations were measured using the TBARS kit from Cayman Chemical Company (Ann Arbor, MI) (28) (link). Samples were thawed and diluted to 10 µg protein/mL in RIPA buffer. One hundred µL of sample or standard was placed in a 5 mL vial. One hundred µL of SDS solution and 4 mL of the color reagent were added to each vial. The vials were placed in boiling water for 1 h, and then immediately placed in an ice bath for 10 min. The samples were centrifuged at 1600 × g at 4oC for 10 min and allowed to stabilize at room temperature for 30 min. Fluorescence at an excitation wavelength of 530 nm and an emission wavelength of 550 nm was determined on duplicate samples and was quantified by interpolation from a standard curve constructed from MDA diluted in RIPA buffer and carried through the assay in parallel.
4
Quantification of Liver Lipid Peroxidation
5
Gastric PGE2 and Lipid Peroxidation
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Gastric levels of PGE2 were detected using the Cayman PGE2 monoclonal enzyme immunoassay kit. In addition, to estimate the grade of lipid peroxidation in the gastric mucous membrane, the level of MDA was measured using Cayman TBARS kit.
6
Lipid Peroxidation Assessment Protocol
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MDA is generated by the degradation of polyunsaturated lipids, used as a biomarker for measuring the lipid peroxidation. The measurement of substance reactive with thiobarbituric acid is a well-established way to screen and monitor lipid peroxidation. Hippocampus and serum MDA were measured by thiobarbituric acid reactive substances (TBARS) kit (Cayman, Ann Arbor, MI, USA). Hippocampus and serum NO concentration was measured using the Griess Reagent kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturers’ protocols.
7
Oxidative Stress Evaluation in Spinal Cord
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In portions of spinal cord, oxidative stress indicators were evaluated: malondialdehyde (MDA) levels via thiobarbituric acid reactive substances (TBARS) kit (Cayman, Chemical Company, Ann Arbor, MI, United States Cat. No. 10009055); DNA oxidation by 8-OHdG immunohistochemistry. Paraffin sections of spinal cord were processed for 8-OHdG immunohistochemistry, using monoclonal antibodies as detailed Table 1 . After deparaffinization and rehydratation, sections were incubated in a blocking solution of bovine serum albumin (BSA) in phosphate buffer saline (PBS) 0.1 M pH 7.4 for 1 h at room temperature. Incubation with primary antibodywas performed over night at 4°Cat condition detailed in Table 1 . After three washes in PBS, sections were incubated in a biotinylated secondary antibody solution (Table 1 ).
8
Quantifying Oxidative Stress Biomarkers
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Oxidative stress was assessed via changes in malondialdehyde (MDA) (Nielsen et al., 1997 (link)). Plasma MDA was quantified by using the TBARS kit (Cayman Chemical Company, Ann Harbor, MI, United States). The kit uses the thiobarbituric acid (TBA) to react and adduct the MDA to form the MDA-TBA under high temperature (90–100°C) and acidic conditions. Briefly, 100 μl of plasma was mixed with 100 μl of SDS solution and 4 ml color reagent containing TBA, acetic acid, and sodium hydroxide. The samples were boiled in a water bath for 1 h, cooled, and the absorbance was measured at 530 nm using fluorometric plate reader. The concentration of MDA was calculated and expressed as μM. In addition, SOD was analyzed as an antioxidant marker protein (Castro and Freeman, 2001 (link)). The plasma total SOD activity (U/ml) was determined by SOD assay kit (Cayman Chemical Company, Ann Arbor, MI, United States) following the manufacturer’s protocol.
9
TBARS Assay for Oxidative Stress
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MDA assays were performed employing a TBARS Kit (Cayman, MI, USA). Assays were performed following the manufacturer’s protocol and absorbance was measured at 540 nm.
10
Quantifying Lung Oxidative Stress
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The malondialdehyde (MDA) concentration was measured using a specific enzyme immunoassay thiobarbituric acid reactive substances (TBARS) kit (Cayman Chemical, Ann Arbor, MI, United States) Briefly, 25 mg of fresh lung tissue were homogenized in RIPA Buffer Concentrate (Cayman Chemical, Ann Arbor, MI United States) and centrifuged at 1600 x g for 10 min at 4°C. The supernatant were collected for analysis. Blood samples were collected from the left ventricle using a syringe with heparin. Blood samples were centrifuged at 1000 x g for 10 min at 4°C and the fresh plasma (supernatant) were use for analysis.
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