2.7. Measurement of cyclooxygenase activity

Cyclooxygenase activity was measured using a fluorometric assay kit (Cayman Chemical Cat No: 700200, Cat Ann Arbor, MI, USA) according to manufacturer’s instructions. The analysis uses the peroxidase component of COX. The reaction between hydroperoxy endoperoxide (PGG₂) and ADPH (10-acetyl-3,7 dihydroxyphenoxazine) resulting from peroxidase COX activity produces highly fluorescent resorufine. Resorufine fluorescence is analyzed at 530 nm excitation and 585 nm emission wavelengths. HEK-293 cells (4–5 × 10⁶) were sonicated in ice-cold PBS containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Cellular lysates were centrifuged at 10,000 g for 15 minutes at 4 °C and supernatants were kept at −80 °C until assayed. One unit of enzyme activity was defined as the amount of enzyme that caused the formation of 1 nmol of fluorophore per minute at 22 °C. Total COX activity in the samples was reported as nmol/min/mg protein.