Cell Culture
Mouse neuroblastoma cells (N2a cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin–streptomycin (Invitrogen, USA), and maintained in a 5% humidified CO2 and 95% air at 37 °C. 1×104 N2a cells/well were seeded onto a 6-well plate until the cells adhered to the well. When the cell grew to 70 to 80 percent, 10μmol/L COX-2 selective inhibitor Celecoxib (Selleck, USA) was added. Then the cell were incubated for 24hour until the culture medium was collected to measure the mRNA of COX-2 by RT-qPCR. Celecoxib was added for 24h in the cell culture medium before stimulation with PGE2 (1μm; Selleck, USA) for 4 hours, then the culture medium was collected to measure the expression of proteins by western blot.