PGE2 and TNF-α expression levels determined by ELISA

The microplate was coated with specific antibodies provided in the ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), incubated for 1 h at 37°C and subsequently at 4°C overnight. The plate was washed three times and 200 µl blocking buffer was added and incubated for 1 h at 37°C. After washing 3 times, cell medium of each group was added and incubated at 37°C for 2 h, and washed three times again. Anti-human immunoglobulin G labeled with horseradish peroxidase was added and incubated at 37°C for 1 h. Stop buffer was added following 3 washes with PBS and 2 with distilled water. The optical density was measured at a wavelength of 450 nm with a microplate reader (model ELX800; BioTek Instrument Inc., Winooski, VT, USA.