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. 2024 Mar 29;7:386. doi: 10.1038/s42003-024-06074-z

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Schematic of the secondary NETosis assay in nanowells where naïve cells were incubated with cells pre-stimulated with ionomycin (6 µM) in clean media. The secondary NETosis was quantified by the measurement of NETs release in naïve cells: The NETs release was evaluated by Hoechst staining of naïve cells and measured as the percentage of cells undergoing NETosis. NETs release by (b) dHL-60 cells and (c) primary neutrophils after co-incubating with ionomycin-stimulated cells at varying cell densities. d Secondary NETosis in primary neutrophils after different treatments on stimulated cells: stimulated cells (no treatment), cell-free NETs isolation, ODA-A151 treatment, DNase I treatment, Proteinase K treatment. All statistical analyses were carried out using one-way ANOVA with Tukey’s post-hoc test *p < 0.05; **p < 0.01; ***p < 0.001. All the values shown were averaged from three individual experiments. Error bars represent standard deviation.