| Assay Method Information | |
| | mPGES-1 Inhibitory Activity Test |
| Description: | mPGES-1 microsomal specimens were prepared by transiently transfecting CHO-K1 cells (DS Pharma Biomedical Co. Ltd) with a plasmid encoding human mPGES-1 cDNA using FuGENE 6 Transfection Reagent (Promega). The prepared mPGES-1 microsome specimens were diluted in potassium phosphate buffer of pH 7.4 comprising 2.5 mM reduced glutathione (Sigma-Aldrich), DMSO solution of a test compound or DMSO was added to a final concentration of 1% of DMSO, and then incubated at 4° C. for 20 minutes. The enzymatic reaction was then initiated by adding a solution of PGH2 (Cayman chemical) substrate of a final concentration 1 μM and incubated at 4° C. for 60 seconds. The reaction was terminated by adding a solution of ferric chloride (STREM CHEMICALS) and citric acid (Wako Pure Chemical Industries) salt to a final concentration of 5 mg/mL and 250 mM, respectively. The formed PGE2 was quantified using the HTRF kit (Cisbio International). Solutions without the test compound were used as positive controls, and solutions without the test compound and microsomal specimens were used as negative controls. 100% activity was defined as production of PGE2 in the positive control minus that in the negative control. The percentage inhibition of production of PGE2 at final concentrations of 10 nM and 100 nM of the test compound was then calculated, or IC50 values were determined using standard methods. |
| Affinity data for this assay | |
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