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.