Oxiselect thiobarbituric acid reactive substances tbars assay kit

Lipid peroxidation in the liver was assessed using the OxiSelect™ thiobarbituric acid reactive substances (TBARS) assay kit according to the manufacturer’s instructions (Cell Biolabs, San Diego, CA, USA). Briefly, approximately 100 mg of tissue was suspended in phosphate buffered saline (PBS) containing 0.05% butylated hydroxytoluene solution (to prevent tissue oxidation) and homogenised at 25 Hz using a Tissue lyser and pre-cooled adapters (Qiagen), by alternating between 1 min in the Tissue lyser and 1 min on ice, and repeated 3 times, followed by centrifugation at 10,000 × g for 5 min at 4 °C to collect the supernatant. The malondialdehyde (MDA) content, a marker of lipid peroxidation, was quantified at 490 nm using a BioTek^® ELx800 plate reader equipped with Gen 5^® software (BioTek Instruments Inc., Winooski, VT, USA). The MDA content was normalised to protein content, quantified using the Bradford reagent (BioRad), and results were expressed as µmol MDA/mg protein.

Malondialdehyde content in leaf samples was measured using an OxiSelect thiobarbituric acid reactive substances (TBARS) assay kit (Cell Biolabs, San Diego, CA, USA, product number STA 330) as an estimate of lipid peroxidation. The TBARS assay is based on the reactivity of MDA with two molecules of thiobarbituric acid (TBA) via an acid-catalyzed nucleophilic-addition reaction. The resulting pinkish-red fluorescent MDA:TBA (1:2) adduct has an absorbance maximum at 532 nm and can be measured colorimetrically [54 (link)]. In the present study, the estimation of MDA content was done as explained by Narayanan et al. [25 (link)].

Approximately 3 mL of blood per hen (one hen per replicate) was drawn from the wing vein into a clot activator tube (Becton Dickinson Vacutainer, Franklin Lakes, NJ, USA) at 4, 8, and 12 weeks. Serum samples were obtained by gentle centrifugation (200× g) for 15 min and stored at −20 °C before analysis. Serum samples were used to measure various biomarkers of oxidative stress, including levels of glutathione peroxidase (GPX), superoxide dismutase (SOD), total antioxidant capacity (TAC), catalase (CAT), MDA, and 8-hydroxydeoxyguanosine (8-OHdG). The level of activity of GPX in serum samples was determined using a GSH-Px ELISA kit (EnzyChrom GPx, BioAssay Systems, Hayward, CA, USA). SOD was analyzed using a SOD determination assay kit (Sigma, St. Louis, MO, USA) and expressed as SOD activity (% inhibition rate). TAC was analyzed using a QuantiChrom antioxidant assay kit (BioAssay Systems, Hayward, CA, USA) and expressed as mmol Trolox equivalents. CAT was analyzed using an OxiSelect catalase activity assay kit (Cell Biolabs, Inc., San Diego, CA, USA). MDA was measured using an OxiSelect thiobarbituric acid reactive substances (TBARS) assay kit (Cell Biolabs, Inc., San Diego, CA, USA). 8-OHdG was determined based on oxidative DNA damage (Cell Biolabs, Inc., San Diego, CA, USA) and expressed in ng/mL.