E bc k020 m

ROS production was measured using an ROS assay kit (E004-1-1, Nanjing Jiangcheng, China). After the cells in the 6-well plates were treated with different concentrations of metformin, the cells were washed with PBS for three times. Subsequently, 1mL of serum free medium containing 10 µM of 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was added into each well and the cells were incubated at 37 °C for 30 min. The six-well plates were imaged by fluorescence microscopy. Then the cells resuspended with 1mL PBS were transferred to 1.5mL EP tube, and were resuspended and washed twice with PBS, and finally resuspended with 200 µL PBS. The cell suspensions were transferred to a 96-well black-walled plate. Fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 520 nm.
The contents of MDA, GSH, and T-SOD, CAT in cells treated with different concentration of metformin were measured using MDA assay kit (A003-4-1, Nanjing Jiangcheng, China), GSH assay kit (A003-2-1, Nanjing Jiangcheng, China), T-SOD activity assay kit (E-BC-K020-M, Elabscience, China), CAT activity assay kit (E-BC-K031-M, Elabscience, China) according to the manufacturer’s instructions, respectively. All experiments were repeated at least three times.

SOD activity was measured using a colorimetric assay kit from Elabscience (E-BC-K020-M). The measurement was carried out using the water-soluble tetrazolium salts-1(WST-1) principal method. MDA levels were assessed using a colorimetric assay kit from Elabscience (E-BC-K025-S) and a modified spectrophotometric thiobarbituric acid (TBA) test method.

SOD enzymatic activity and ATP levels in NP cells were detected using a Total Superoxide Dismutase (T-SOD) Colorimetric Assay Kit (WST-1 method) (Elabscience: E-BC-K020-M) and Adenosine Triphosphate (ATP) Colorimetric Assay Kit (E-BC-K157-M), respectively, in the light of the manufacturer's instruction. Human NP cells were fostered at 37°C in 5% CO₂ after being treated. Briefly, the cells were scraped off with a scraper and collected under the centrifugation at 1000 × g at 4°C for 10 minutes. Then, the 10⁶ cells were lysed with 300 μl-500 μl of homogenate medium and centrifuged. The supernate was collected and measured by adding the relevant test working fluid.