Measurement of H2O2 production
The supernatants of stimulated peritoneal murine macrophages cultures
were collected and analyzed for hydrogen peroxide
(H2O2) concentrations by using a
colorimetric assay adapted to the laboratory
conditions36,39. Briefly, 50 μL supernatants from each
culture were placed into a 96-well plate and mixed an equal volume of
fresh phenol red solution (5.5 mM dextrose, 0.056 g phenol red and 8.5
U/mL Type I HRP in DPBS) followed by incubation at room temperature and
protected from light for 3 h. Afterwards, the reaction was stopped by
adding NaOH solution, and absorbance was measured at 490 nm using a
microplate absorbance reader (Thermo Scientific, Multiskan FC). Hydrogen
peroxide concentration was determined based on a
H2O2 standard curve ranging from 0 to 50
μM/mL. All experimental variants were performed in triplicate.