Nitric Oxide Griess Assay

Nitric oxide was measured in the splenocyte culture supernatant by Griess assay (21). Eight weeks after the challenge with L. major^EGFP, mice were sacrificed and splenocytes (1 × 10⁶ cells/mL) were restimulated with SLA ^L.major. The cultured cells (48 h) and supernatants (72 h) were collected to measure arginase activity and Nitrite concentration, respectively. The Griess assay determines nitrite concentration as a byproduct of the iNOS enzyme which indirectly indicates NO production. Briefly, Griess reagents including 1% sulfanilamide in 5% H₃PO₄, and NED solution including 0.1% naphthyl ethylene diamine (NED) dihydrochloride in distilled water (DW) were prepared. In a 96-well plate, 50 μL of sulfanilamide solution was added to 50 μL of sample and incubated 5 min in a dark place. Then, 50 μL of NED solution was added and incubated again for 5 min in a dark place. The absorbance was measured at 540 nm. Various concentrations (0–100 μM) of sodium nitrite in RPMI 1640 medium were used as standards to plot a standard curve.