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. 2017 Aug 1;7:7072. doi: 10.1038/s41598-017-07288-4

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© The Author(s) 2017

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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Confocal microscopy showing internalization of OMV-associated DNA in A549 lung epithelial cells. EdU-labeled OMVs, either (a) untreated or (b) DNase-treated, were labeled with the lipophilic stain DiO then added to A549 cells for 5 h, washed to remove non-internalized OMVs, fixed and permeabilized. EdU was detected in a 2-step reaction consisting of incubation with biotin azide, in which the azide binds specifically to the alkyne backbone of EdU, followed by fluorescent detection with streptavidin-conjugated Alexa-Fluor 568. Co-localization of DiO (green) and EdU (red) labels in internalized OMVs are indicated by arrows. Nuclei are stained with DAPI (blue). Merged images showing control samples in which either: (c) biotin azide and streptavidin-AlexaFluor 568 only, (d) no biotin azide or (e) DiO only were added to cells. (f) Non-permeabilized cells. Scale bar = 15 μm.