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. 2018 Jul 31;8:11475. doi: 10.1038/s41598-018-29659-1

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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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Hypoxia delays apoptosis, necrosis and increases neutrophil viability in M.tb infection. (a) The percentage of apoptotic neutrophils (Annexin V positive) is lower in hypoxia than normoxia for uninfected and infected cells. *p < 0.05 between normoxia M.tb 10 and hypoxia M.tb 10 by 2-way ANOVA with Bonferonni post-test. (b) Mean fluorescent intensity of Annexin V positive cells was higher in cells in normoxia than in hypoxia. ****p < 0.001 between normoxia control vs. hypoxia control; normoxia M.tb 1 vs. hypoxia M.tb 1; and normoxia M.tb 10 vs. hypoxia M.tb 10. (c) The proportion of necrotic cells stained positive for Live-Dead dye was lower in hypoxia than normoxia for uninfected and infected cells. ****p < 0.001 between normoxia control vs. hypoxia control; normoxia M.tb 1 vs. hypoxia M.tb 1; and normoxia M.tb 10 vs. hypoxia M.tb 10. (d) Mean fluorescent intensity of Live-Dead dye positive cells was higher in infected and uninfected cells in normoxia than in hypoxia. ****p < 0.001 between normoxia control vs. hypoxia control; normoxia M.tb 1 vs. hypoxia M.tb 1; and normoxia M.tb 10 and hypoxia M.tb 10. (e) The proportion of viable cells is higher in hypoxia than normoxia for uninfected and infected cells, analysed by Annexin V staining. ****p < 0.001 between normoxia control vs. hypoxia control; normoxia M.tb 1 vs hypoxia M.tb 1; and normoxia M.tb 10 vs hypoxia M.tb 10. Bars represent mean ± s.e.m of n = 4 neutrophil donors on 4 separate occasions.