A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture

A simple, rapid and inexpensive method for the measurement of hydrogen peroxide (H2O2) produced by cells in culture is described. The assay is based on the horseradish peroxidase of a (HRPO)-mediated oxidation of phenol red by H2O2 which results in the formation of a compound demonstrating increased absorbance at 610 nm. A linear relationship between absorbance at 610 nm and concentration of H2O2 was found in the 1–60 μM (1–60 nmoles/ml) range. Due to the non-toxic character of phenol red and HRPO, the assay permits measurement of H2O2 production and release by macrophages for time intervals of 5–60 min under regular tissue culture conditions. Using this assay, the ability of a number of agents to induce H2O2 release by guinea pig peritoneal macrophages was demonstrated. These agents were: phorbol myristate acetate (PMA), opsonized zymosan, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and A23187.