Dyadichkina O.V., Korotina O.L., Radetskaya L.E., Generalov I.I.
The possibilities of fluorescence and light microscopy in the detection of extracellular neutrophil traps
Abstract.
Objectives. To compare the reproducibility of the results of neutrophil extracellular traps (NETs) determination using fluorescence microscopy and light microscopy with DNA selective dyes.
Material and methods. We examined neutrophilic granulocytes isolated from the peripheral blood of women with physiological pregnancy in the 2nd and the 3rd trimesters of gestation. Spontaneous (180 minutes incubation with saline) and stimulated (180 minutes incubation with Staphylococcus aureus) ability of neutrophils to form neutrophil extracellular traps was assessed by means of microscopic examination.
Results. Reproducibility results analysis was made after different types of microscopy and fixed smears staining was applied for the determination of spontaneous and stimulated neutrophil extracellular traps production levels. Highly sensitive fluorescent dyes Sytox Green, DAPI, acridine orange and Hoechst 33342 were used. Leukocytes and DNA traps were determined using Hoechst 33342 by means of fluorescence microscopy with appropriate radiation sources and color filters. Examination of stained cells with methyl green dye was carried out by light microscopy. Hoechst 33342 was found to be an optimal fluorescent dye for neutrophil extracellular traps detection. The results obtained by fluorescent microscopy using Hoechst 33342 and light microscopy with methyl green dye were not statistically different.
Conclusion. Assessment of neutrophil extracellular traps by light microscopy with methyl green dye is equivalent to fluorescent microscopy with Hoechst 33342; thus, it can be used in laboratory practice of resource-limited health care settings.
Key words: neutrophil extracellular traps, innate immunity, fluorescent microscopy, light microscopy, Hoechst 33342, methyl green.
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