IMPROVED DETECTION OF NITRIC-OXIDE RADICAL (NO-CENTER-DOT) PRODUCTION IN AN ACTIVATED MACROPHAGE CULTURE WITH A RADICAL SCAVENGER, CARBOXY PTIO, AND GRIESS REAGENT

An improved method for the detection of nitric oxide radicals (NO. in cultures of activated macrophages was developed, involving a nitric oxide radical scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (carboxy PTIO) and Griess reagent. A murine macrophage-like cell line, J774.1, was activated with interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS), which induced the production and secretion of NO; into the culture supernatant. Addition of carboxy PTIO to the activated macrophages increased the amount of NO2-, to 4- to 5-fold without cell damages, probably because carboxy PTIO rapidly reacted with NO. to form NO2-, which was finally assayed by the Griess reaction.