Abstract
A strategy for designing probes based on protection-deprotection chemistry involving fluoresceins and their benzenesulfonyl (BES) derivatives has led to the development of a much more practical superoxide (O(2) (-.)) probe than the previously reported bis(2,4-dinitro-BES) tetrafluorofluorescein (6 a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6 a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7 j) as the optimal reagent. A microtiter plate assay with BESSo showed a tenfold improved detection limit for O(2) (-.) compared with such an assay based on 6 a. BESSo showed markedly better specificity for O(2) (-.) than for GSH or other reactive oxygen species, and this specificity was significantly higher than that of Fe(2+) and some reducing enzymes. These features have resulted in the development of an assay based on BESSo that is capable of providing more unambiguous results for O(2) (-.) release from neutrophils, with or without stimulation by phorbol myristate acetate, as compared with an assay based on 6 a. Intracellular generation of O(2) (-.) in human Jurkat T cells stimulated by butyric acid has been measured by using flow cytometry and fluorescence microscopy utilizing the acetoxymethyl derivative of BESSo.
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