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. 2018 Feb 16;8:3135. doi: 10.1038/s41598-018-21450-6

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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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Evaluation of CD15⁺ cell phenotype. (a–b) PBMC from one day-old healthy donor blood were isolated over Ficoll using conventional tubes (“No membrane, PBMC” and “No membrane, pellet”) or with Accuspin tubes (“Over membrane” and “From membrane”). (a) By light microscopy, a typical neutrophilic appearance was seen. (b) The same cells as in (a) were stained for CD15 and activation markers, CXCR2, CD54 and CD62L. CD15⁺ gated cells are shown, and representative for 7 independent experiments. (c) PBMC from healthy donor blood sample were isolated the same day, or next day, over Ficoll using non-membrane tubes. On the left: non-altered, good viability and typical FSC-SSC properties of neutrophils are shown in pseudocolor plots. Additionally, location of CD15⁺SSC^hi population of neutrophils is shown by backgating (neutrophils are blue). On the right: 2^nd and 4^th rows show the full phenotype of gated CD15⁺SSC^hi neutrophils, while 1^st and 3^rd rows identify the same CD15⁺SSC^hi population (blue) within the same markers on axes, but compared to the staining of all viable cells (backgating). Presented dot plots consist of 23,728 ± 7,040 (Mean ± SEM) events, not including events in the first column of ungated cells, i.e. left panels of (c).