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. 2021 Sep 8;12:702677. doi: 10.3389/fphar.2021.702677

FIGURE 1.

FIGURE 1

(A) Neutrophils isolated from healthy subjects were allowed to adhere on fibrinogen-coated slides in the absence or presence of endotoxin (10 μg/ml) and cultured for 4 or 18 h. At the end of the incubation, samples were fixed and stained for DNA (blue staining, DRAQ-5). (B) Neutrophils from healthy subjects pretreated with CFTRInh-172 (10 µMoles/L) or a vehicle were allowed to adhere on fibrinogen-coated surfaces and stimulated with different agonists for 4 or 18 h. At the end of the incubation, free DNA was quantitated. Briefly, 2.5 µl of endonuclease (nuclease micrococcal, from Staphylococcus aureus 50 U/ml) per 500 µl of the sample was added, and samples were incubated at 37°C for 10 min. The reaction was stopped with 5 µl of EDTA (0.5 M). Samples were centrifuged at 10.000 x g for 3 min, and supernatants were stored at -20°C until DNA quantification by Quant-iT™ dsDNA high-sensitivity assay kit (Invitrogen). Results are means ± SEM of experiments with cells from 4 different donors performed in duplicates.