Measurement of hydrogen peroxide production by phagocytes using homovanillic acid and horseradish peroxidase.

摘要: Publisher Summary This chapter describes a method for the determination of hydrogen peroxide (H 2 O ) produced by neutrophils, monocytes, and macrophages. The assay is based on oxidation homovanillic acid (HVA) into fluorescent dimer, which catalyzed horseradish peroxidase depends H generated cells. Human neutrophils are purified from anticoagulated blood, suspended in isotonic NaCl (10 7 -10 8 cells/ml), kept at 6–10° C until use. Mononuclear phagocytes may be used as suspension or monolayers small plastic culture wells. In both cases, medium Hanks' balanced salt solution (HBSS). Mouse bone marrow-derived macrophages obtained proliferation Teflon bag cultures, harvested bags, centrifuged 200 g 10 minutes room temperature, resuspended desired density 199 containing 15% heat-inactivated horse serum m M glutamine seeding plates HBSS direct monocytes buffy coats donor blood centrifugal elutriation adherent cultures flat-bottom 96-well microtiter plates.