2.4. Nitric Oxide (Griess Reagent Assay)

Cells were seeded at 2 × 10⁴ cells per well in a 96-well plate and incubated at 37 °C with 5% CO₂ until cells reached 80% confluence. Cells were treated for 24 h with control media, 1 µg/mL LPS or both LPS and 300 µM buspirone. The supernatant was collected and placed into a new 96-well plate. A total of 100 µL of freshly prepared Griess reagent was then added to each well and incubated at room temperature for 15 min on a slow oscillation protected from light. Absorbance was measured at 540 nm using the TECAN infinite M1000-PRO ELISA reader. Optical density values from each group were recorded and reported as a percentage of control.