2.6. COX-2 Inhibition Assay

Recombinant human COX-2 (1.25 unit) was incubated with 1 μM hematin, 5 mM l-glutatione, 5 mM dopamine haydrochloride, 5 mM EDTA and different concentrations of inhibitors or vector (DMSO) in 150 μL potassium phosphate buffer (pH 8.0) at 37 °C for 15 min. Then 50 μL AA was introduced into the samples at 10 μM to start the reaction. After incubated 10 min, the samples were put on the ice and stopped by adding 50 μL stop solution, which contains 1% formic acid and 100 ng/mL PGE2-d₄ (internal standard). Before using UPLC-MS/MS to measure the PGE2, the pH of samples was adjusted by 0.5 M NaOH solution to around 8.