E el h6008

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.

For quantification of the two differentially expressed cytokines, IL-8 (Elabscience, E-EL-H6008) and VEGF-A (Elabscience, E-TSEL-H0026) ELISA kits were used according to the manufacturer’s instructions. Standard or CM was added to each coated well and was incubated for 2 h at 37 °C. Then, plates were incubated with the biotinylated antibody for 1 h at 37 °C and then with HRP-avidin for 1 h at 37 °C. After additional washing steps, TMB substrate was added to each well for 15 min at 37 °C, following which, a stop solution was added for color development. The plates were read at 450 nm with an automatic plate reader (Multiskan Sky, Thermofisher, USA). Standard curves were generated using kit-provided standards and were used to calculate cytokine concentrations in the CM.