E bc k031 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.

The skin homogenate was prepared by homogenizing tissue with phosphate-buffered saline at pH 7.4. We used commercially available kits to determine the levels of superoxide dismutase (SOD) (Cayman, Ann Arbor, MI, USA) and catalase (CAT) (#E-BC-K031-M, Elabscience, Cayman, Ann Arbor, MI, USA). The hearts of the mice were punctured to obtain blood samples, and plasma was separated. The plasma was spun at 10,000 rpm for 5 min at room temperature, and the supernatant was aspirated. Malondialdehyde (MDA) levels were measured using commercially available assays (MDA # E4601-100, Biovision, San Francisco, CA, USA).

The activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) were measured using GSH-Px, CAT, and SOD activity assay kits (Catalog No.: E-BC-K096-M, E-BC-K031-M, E-BC-K020-M; Elabscience, Wuhan, China), respectively. Malondialdehyde (MDA), interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) levels were measured using MDA, IL-1β, IL-6, IL-8, and TNF-α ELISA kits (Catalog No.: E-EL-0060c, E-EL-H0149c, E-EL-H6156, E-EL-H6008, E-EL-H0109c, Elabscience). After cell culture at 24 h, the culture supernatant was collected and centrifuged at 1000 × g for 20 min to remove impurities and cellular debris. The supernatant was collected for further analysis. For the detection, 50 µl of the supernatant was taken and analyzed. Following completion of the experiment, the optical density (OD) of each well was measured using a Multiscan MK3 reader (Thermo Fisher Scientific). All reactions were repeated thrice.