Quantification of lipid peroxidation using TBARS assay

Parameter™ TBARS Assay kit (R&D Systems, Minneapolis, MN, USA) was used to quantify lipid peroxidation by measuring TBARS levels, as an indicator of oxidative stress in cells. The assay was performed in SH-SY5Y and T98G cells after PA treatment, according to the manufacturer’s protocol. Briefly, cell lysate was prepared by first collecting the cells and washing the cells with cold PBS. Then, the cells were resuspended in deionised water at the density of 1 × 10⁶ cells/ml. The cell suspension was subjected to a total of 3 cycles of 10-s sonication and then freeze/thaw at ≤−20 °C. Then, 300 µl of the cell lysate was subjected to acid treatment with 300 µl of TBARS Acid Reagent provided in the kit. After 15 min of incubation at room temperature, the mixture was centrifuged at ≥12,000 g for 4 min and the supernatant was retained. Next, 150 µl of standards and samples were added into each well of the microplate and 75 µl of TBA Reagent was added. The optical density of each well was pre-read at 532 nm using FLUOstar® Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany). Then, the microplate was covered with the adhesive strip provided and was incubated at 45–50 °C for 2–3 h. After incubation, the optical density was read at 523 nm. The pre-reading was subtracted from the final reading to correct for the samples contribution to the final absorption at 532 nm. The results were calculated according to the manufacturer’s instruction. A linear standard curve was plotted and the concentrations of samples were read from the standard curve and were multiplied by the dilution factor 2.