Lipid peroxidation assay

Lipid peroxidation was measured based on the production of thiobarbituric acid reactive substances (TBARS). Cells in 96-well plates (2 × 10⁴ cells/well) were pre-incubated with glucose (5.5 and 30 mM) for 48 h at 37 °C and 5% CO₂. Next, the cells, with or without 10, 25, 50, and 100 μg/mL SSE, were further incubated for 24 h. A sample of 200 μL was mixed with 400 μL of the TBARS solution and boiled at 95 °C for 20 min. The absorbance at 532 nm was measured, and the TBARS concentrations were extrapolated from the 1,1,3,3-tetraethoxypropane standard curve. The thiobarbituric acid value was expressed as the equivalent nanomoles of malondialdehyde (MDA).