5.6. Griess Reagent Assay

Griess assay is a colorimetric technique that measures nitrite (NO₂‐), one of the two primary stable forms of nitric oxide. This assay relies on the reaction described in 1879 by Griess [16]. In order to assess the effects of lithium on nitric oxide (NO) production, cells were seeded in 96-well plates at a density of 6 × 10⁶ cell/well in a phenol-free medium overnight. Cells were pretreated for 1 hour with various concentrations of lithium (5 and 10 mM) or 10 mM NaCl and activated with 10 μg/ml LPS (Sigma, USA) for 24 hr. After 24 hr of treatment, nitric oxide was measured by mixing equal amounts of phenol-free medium and Griess reagent (Sigma-Aldrich, USA). The absorbance was measured using GloMax-Multi microplate reader at 550 nm (Promega, USA).