Measurement of NO production
Supernatants of stimulated peritoneal murine macrophages cultures were
collected and analysed for nitrite production as an indicator of nitric
oxide (NO) synthesis by using the Griess reagent37,38.
Briefly, 50 µL of supernatants from each culture were placed into a
96-well plate and mixed with an equal volume of Griess reagent (0.1%
N-(1-naphthyl) ethylenediamine dihydrochloride in H2O
and 1% sulfanilamide in 5% H3PO4),
followed by incubation at room temperature and protected from light for
15 min. Consecutively, the absorbance was measured at 490 nm using a
microplate absorbance reader (Thermo Scientific, Multiskan FC). Nitrite
concentration was determined based on a sodium nitrite
(NaNO2) standard curve ranging from 0 to 50 μM/mL. All
experimental variants were performed in triplicate.