Serum and Supernatant 8-OHdG Measurement

Serum or culture supernatants 8-OHdG levels were measured by human 8-hydroxydeoxyguanosine ELISA kit (CUSABIO, Wuhan, China) according to the manufacturer’s instruction. This assay employs the competitive inhibition enzyme immunoassay technique. In brief, samples were thawed at room temperature and centrifuged at 13.4×g for 3 minutes before use. Then, 50 μL of the standard or a sample per well was added into each well of a 96-well microplate that had been coated with the 8-OHdG-specific antibody; 50 μL of horseradish peroxidase (HRP) conjugate was also added to each well. The mixtures were combined well and incubated at 37 for 60 minutes. After aspirating and washing each well 3 times with 200 μL of wash buffer (1×) per well, 50 μL of substrate A and 50 μL of substrate B were added to each well. Incubation at 37 for 15 minutes in humid and dark incubator followed. Then, 50 μL of stop solution was added to each well and the optical density of each well was determined within 10 minutes, using a microplate reader (Autobio, Zhengzhou, China) set to 450 nm, with the correction wavelength set to 600 to 630 nm. The detection range was 2 to 800 ng/mL. The assay was performed in technical duplicate.