Mda detection kit

A piece of testicular tissue from each mouse in each of the four groups was immediately homogenized in ice-cold PBS. Total protein extraction and protein concentration measurements were performed as described above to analyze the enzyme activities. The T-AOC of testicular tissue was measured by the rapid 3-ethylbenzthiazoline-6-sulfonic acid method (cat# S0121, Beyotime). Total SOD activity and GPx activity in testicular tissue were measured with a total superoxide dismutase assay kit (cat# S0109, Beyotime) with nitroblue tetrazolium (NBT) and a glutathione peroxidase assay kit (cat# S0056, Beyotime) with nicotinamide adenine dinucleotide phosphate (NADPH), respectively. The levels of hydrogen peroxide and MDA in testicular tissue were detected using a hydrogen peroxide detection kit (cat# S0038, Beyotime) and an MDA detection kit (cat# S0131, Beyotime) based on the chromogenic reaction between MDA and thiobarbituric acid (TBA).23 (link)24 (link)25 (link)26 (link) The T-AOC, SOD, GPx, hydrogen peroxide, and MDA levels were normalized to that of the total protein. Five or six mice from each group were used in these experiments to measure the level of oxidative stress in testicular tissue.

The T-AOC was measured by the rapid 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) method (#S0121, Beyotime, China). The peroxidase substrate used in this method is 2,2′-azino-bis ABTS, which produces a soluble end product that is green in color and can be read spectrophotometrically at 405 nm. The presence of antioxidant molecules inhibits production of the colored product, thereby decreasing the absorbance at 405 nm. Trolox was used as a standard antioxidant reagent to generate a standard curve. Lens capsules and cultured LECs were used in this assay. The amount of MDA in lens capsules was detected using an MDA detection kit (#S0121, Beyotime, China) based on the chromogenic reaction of MDA and thiobarbituric acid (TBA). The absorbance of the MDA-TBA adduct was measured at 535 nm. Lens capsule samples were used in the MDA assay. The experiments were conducted according to the manufacturer's instructions.

SGC7901 and BGC823 cells were cotreated with erastin (5 mmol/L) or ferrostatin (1 mmol/L). After 48 h, the cell viability was analyzed by MTT assay. Elevated iron level and accumulated lipid reactive oxygen species (ROS) were representative characteristics of ferroptosis. We used an iron assay kit (Beyotime, China) to examine the level of intracellular Fe²⁺. The cells were homogenized to collect the supernatant, incubated with iron reducer, followed by labeling with iron probe. OD 590 nm was detected in a microplate reader (PerkinElmer, Waltham, MA, United States). For detection of lipid ROS, cells were stained with BODIPY C-11 dye (Beyotime) for 30 min, and subsequently detected by flow cytometry (BD Biosciences, Franklin Lakes, NJ, United States). The levels of malondialdehyde (MDA) and glutathione peroxidase (GSH) was measured by the MDA detection kit (Beyotime) and GSH assay kit (Cayman, Ann Arbor, MI, United States), respectively.