A sensitive flow cytometric method for measuring the oxidative burst

We report a novel method of flow cytometric detection of the oxidative burst in human neutrophils. The cells were covalently labeled on the plasma membrane by incubation with 4-carboxydihydrotetramethylrosamine succinimidyl ester on ice. Activation of neutrophils resulted in an increase in red fluorescence at 575 nm using 514 nm excitation. The sensitivity of this assay in detecting the response to chemotactic peptide N-formyl-met-leu-phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) was comparable to that observed with the cytochrome c oxidation test. We compared the new method to previously described methods using intracellular fluorescent stains. Our method showed the lowest nonspecific oxidation and the best sensitivity to FMLP-induced activation. It was equally or slightly less efficient than the green fluorescent stain dihydrorhodamine 123 in detection of neutrophil activation by immune complexes and PMA but much more sensitive than red fluorescent stains hydroethidine and dihydrotetramethylrosamine. It can be successfully used in kinetic experiments and yields a fluorescence signal that remains stable for at least 1.5 h after formaldehyde fixation with antioxidant added.