HOCl образуется при стимуляции нейтрофилов благодаря активности миелопероксидазы. Для селективной спектрофотометрической детекции HOCl предложен целестиновый синий B (CB). Целью данной работы явилась разработка флуоресцентных методов оценки продукции HOCl нейтрофилами. Продукт реакции CB с HOCl флуоресцировал при 578 нм (возбуждение 487 нм). Методом конфокальной микроскопии получили изображения активированных нейтрофилов, окрашенных CB. При проточной цитометрии флуоресценция активированных 50 нМ PMA нейтрофилов была в 5 раз выше (p < 0,05), чем у клеток без активатора.
Hypochlorous acid (HOCl) is produced with myeloperoxidase (MPO) in activated neutrophils. To assay MPO activity we chose a HOCl-selective dye i.e. celestine blue B (CB) that changes into pink glycol after oxidation. Our aim was to elaborate fluorescent methods of measuring HOCl production with activated neutrophils. Scanning CB fluorescence before and after its reaction with HOCl revealed activation and maximum emission, at 487 and 578 nm respectively, which are specific for oxidized product. Activated with Phorbol 12-myristate 13-acetate (PMA), the neutrophils were incubated with MPO inhibitor, 4-aminobenzoic acid hydrazide, which decreased fluorescence intensity (activation 487 nm, emission 578 nm) as compared with inhibitor-free samples. By confocal microscopy method we obtained images of CB- and DAPI-stained neutrophils. Neutrophil extracellular traps (NET) are clearly visible: DAP1 staining of multiple DNA bands co-localizes with fluorescence of oxidized CB. Flow cytometry showed that intensity of neutrophils activated by 50 nM PMA was 5 times higher (p < 0.05) than in PMA-free cells.
The authors declare that there are no conflicts of interest present.