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. 2018 Apr 30;8:6777. doi: 10.1038/s41598-018-25167-4

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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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Schematic diagram of neutrophil migration model. Diagram of cell seeding, A549 infection (A) and neutrophil migration (B). (1) A549 cells were seeded onto the underside of a transwell and allowed to attach for 4 hours. (2) Transwells were subsequently inverted and maintained in media to allow a confluent epithelial monolayer to develop for 72 hours. (3) Transwell were inverted and infected with GFP RSV or mock infected for 2 hours. (4) Transwells were inverted and maintained in media and infection allowed to progress for 24, 48, 72 or 96 hours. (5) Ultrapure neutrophils were added to the basolateral side of the transwell, and were allowed to migrate for 1 or 4 hours. Underneath the transwell was HBSS+ or RSV infection media. (6) Post migration basolateral and apical neutrophils were collected and downstream analysis performed.