2.9. Cyclic Adenosine Monophosphate (cAMP) Assay

Cellular cAMP production was measured using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) [32, 33]. Cells were seeded into 6-well plates at a density of 5 × 10⁵ cells/well. The media were aspirated 20 or 120 minutes after exposure to test substances, and 250 μl of 0.1 N HCl was introduced. After 20 minutes incubation at room temperature, cells were scraped and centrifuged. The supernatants were stored at -80°C until the time of measurement. For the experiment with 120-minute exposure to test substances, the medium contained 0.5 mM 3-isobutyl-1-methyl xanthine (IBMX), a phosphodiesterase inhibitor, to inhibit cAMP degradation.