Mda assay kit

The cell supernatant or serum of each subgroup was collected after centrifugation (1,000 ×g, 10 minutes) to analyze the levels of nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH). The following kits were used according to the manufacturer’s instructions: NO content assay kit (BC1475, Solarbio), MDA assay kit (BC0025, Solarbio), GSH assay kit (BC1195, Solarbio), and SOD activity detection kit (BC5160, Solarbio).

The cell ferrous iron colorimetric assay kit and MDA assay kit from Solarbio (BC5315&BC0025, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) were used for determining the intercellular ferrous iron and the level of MDA in the PC cells. Following the manufacturer's instructions, we added 0.5 ml extracting solution into 5 × 10⁶ cells. Then, the cells were crushed by ultrasonic in an ice bath and subsequently centrifuged at 4 °C at 8000 g for 10 min to acquire the supernatant. Afterward, Multiskan™ GO Spectrophotometer (Thermo Scientific, USA) was preheated to 25 °C for 30 min. Subsequently the supernatant was incubated with reagent mixture at 25 °C for 10 min, and then the absorbance value at 510 nm was detected to calculate the intracellular ferrous iron level. As for the level of MDA, the mixture of the obtained supernatant and the reagent was kept in a water bath at 100 °C for 60 min and cooled in an ice bath immediately after. Then, the samples were centrifuged at 10,000 g for 10 min at room temperature, 200μL of the above supernatant was plated in 96-well microplate and the absorbance at 532 nm and 600 nm of each sample was measured to calculated MDA level.