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. 2018 Sep 10;8:13490. doi: 10.1038/s41598-018-31391-9

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© The Author(s) 2018

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Live cell laser-scanning confocal microscopy of human PMN following the addition of the probe (8) (5 µM). Non-activated neutrophils are seen in row A. Rows B and C depict neutrophils that have been activated by co-culture with Pseudomonas aeruginosa (J3284). Row C neutrophils were additionally co-cultured in the presence of Sivelestat (100 µM). Column 1: Extensive vesicular formation is seen in neutrophils stained with Calcein-AM. Probe (8) fluorescence (green) signal (column 2) is seen in activated neutrophils but is absent in quiescent neutrophils or in the presence of Sivelestat. The merged images (column 4) show the co-localisation of labelled bacteria (PKH) and probe (8) signal within a neutrophil vesicle. Far right panel shows magnified image of co-localised bacteria and probe (8) signal. Scale bar: 5 µm. Images are representative of fields of view from three independent experiments.