Mda lipid peroxidation microplate assay

To assay lipid peroxidation, the MDA lipid peroxidation microplate assay (Merck KGaA) was used according to manufacturers' instructions. Briefly, cells were homogenized in lysis solution containing butylated hydroxytoluene. The insoluble fraction was removed by centrifugation, and the supernatant was used for analysis. The supernatants were mixed with thiobarbituric acid solution reconstituted in glacial acetic acid and then incubated at 95 °C for 60 min. The supernatants containing MDA-thiobarbituric acid adduct were added into a 96-well microplate for analysis. A microplate reader was used to measure the absorbance at OD 532 nm.
For glutathione determination, intracellular reduced form glutathione (GSH) and its oxidized form (GSSG) were assessed using a GSH/GSSG Ratio Detection Assay Kit II (Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, cells were homogenized in 5% 5-sulfosalicylic acid, and the insoluble fraction was removed by centrifugation. The resultant supernatant was added to double-deionized H₂O (ddH₂O) to reduce the 5-sulfosalicylic acid concentration to 0.5% for the assay. A microplate reader was used to measure absorbance at OD 415 nm. The concentration of GSH was calculated by subtracting 2 × GSSG from the total glutathione concentration.