Measurement of Serum Nitric Oxide and PGE2 Production

On the day of sacrifice, whole blood samples were collected and the blood was allowed to coagulate for 30 min. Then, the serum was separated by centrifugation for 10 min at 2,000 rpm at 4 °C and stored at ‑80 ˚C until further use. Productions of NO in the serum samples were determined using the Griess reaction for nitric oxides. Briefly, 100 µl serum was mixed with 50 µl Griess reagent A and 50 µl Griess reagent B, followed by incubation for 15 min at 37 ˚C. Optical density was measured at 540 nm on an ELISA reader. Productions of PGE2 in the serum samples were determined using an ELISA kit (R&D Systems), per the manufacturer's instructions.