The nitric oxide (NO) production in cell culture were determined
by measuring the nitrite (NO2) formation by using Griess assay
as described previously (Kim et al., 2008). Briefly, 100 lL of
griess reagent (Sigma–Aldrich, USA) containing 1% sulfanilamide
and 0.1% naphtylethyenediamine dihydrochloride in 2.5% H3PO4
was added to 100 lL of cell culture supernatant and incubate for
15 min at room temperature in the dark. The colour density was
measured at 540 nm using a microplate reader (Molecular Devices,
USA). A standard curve was generated in the same method using
NaNO2.