Experience in development and application of a technique for studying the ability of neutrophils to form extracellular traps

The phenomenon of neutrophil extracellular trap (NETs) formation, or netosis, plays a key role in pathogenesis of infectious and inflammatory diseases. However, the lack of standardized methods for assessing NETs makes it difficult to correctly interpret the results. The aim of our study was to develop a feasible methodology to investigate the ability of neutrophils to form NETs, which would allow precise identification of netosis-associated phenomena. Neutrophils were isolated from peripheral blood of 15 healthy donors by gradient centrifugation (Ficoll-Verographin, density gradient 1.077-1.105 g/mL). A mixture of bacteria (Lactobacillus reuteri, L. acidophilus, L. rhamnosus, Bifidobacterium longum) was used to stimulate netosis. Propidium iodide (PI), a DNA-intercalating dye, and murine monoclonal antibodies to human CD15/FITC were used to visualize NETs and other fluorescent objects. Luminescence microscopy was performed at excitation wavelengths of 450-480 nm and emission wavelengths of 515 nm. We have registered the following morphological variants: (1) intact neutrophils (bright green, without nuclear staining); (2) activated neutrophils (bright green with a red-orange, PI-stained nucleus); (3) early netosis cells (bright green cells with nuclear material released to one or more sites at cell membrane); (4) NETs: (a) cloud-shaped objects surrounding the neutrophil or its fragments from all sides, being and more than 1.5 times its size, and (b) filamentous forms seen as filament structures exceeding the neutrophil size>2-fold or more. The proportion of each type of observed objects relative to all fluorescent-positive objects was determined. Upon optimization of this method, the key parameters were determined: temperature of cell incubation with the stimulant (37 °C); its concentration (2.5x10⁹ bacteria/ mL), stimulation time (30 min). Testing the reproducibility of this technique showed an acceptable inter-series coefficient of variation (CV): 13.8% for filamentous and 15.2% for cloud-shaped NETs. The proposed method was applied in different groups of patients with certain infectious (pulmonary tuberculosis) and non-infectious pathology (ulcerative colitis, aneurysmal bone cysts). Testing the NET-producing ability of neutrophils in the subjects from these cohorts allows us to conclude that the levels of sensitivity and manifestations for these indexes registered with this technique are quite satisfactory.

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