Quantifying Lipid Peroxidation via TBARS Assay

We performed the TBARS assay to quantify the lipid peroxidation marker malondialdehyde (MDA), according to the method reported by Zhang and Huang [16 ], with minor modifications. Briefly, 10 mL of cell culture was centrifuged for 10 min at 10,000× g. The pellet was then mixed in a tube with 1 mL 0.1% trichloroacetic acid (TCA), and placed in an ultra-sonic bath at room temperature for 3 min to promote extraction. Next, the tube was centrifuged (10,000× g for 10 min at 4 °C) and the supernatant was transferred to a new tube. A 200 µL aliquot of supernatant was mixed well with 1 mL 20% TCA containing 0.5% TBA. This mixture was boiled at 95 °C for 15 min, quickly cooled on ice, centrifuged at 10,000× g for 5 min, and then the supernatant was transferred to a cuvette. Finally, we measured the absorbance at 532 nm using a PerkinElmer Lambda 12 spectrophotometer (PerkinElmer, Shelton, CT, USA). A molar extinction coefficient of 155 cm⁻¹ mM⁻¹ was used for MDA determination. The results were corrected for the experimental blank, and the MDA concentration was calculated as described by Lin et al. [17 (link)].