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. 2019 Aug 5;9:11321. doi: 10.1038/s41598-019-47475-z

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© The Author(s) 2019

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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Under-medium module exchange procedure. (a) The glass slide on which the two endothelial cell populations were seeded was submerged in a Petri dish containing cell medium and positioned in the alignment tool. (b) The flow module was connected to a vacuum source, and the outlet is connected to a syringe containing culture medium. A reservoir for leukocyte loading was inserted in the inlet. (c) The flow module was lowered into the Petri dish containing the endothelial cell slide positioned in the alignment tool, vacuum was applied and the perfusion chamber and leukocyte reservoir was filled with cell culture medium via the outlet tubing, to confirm that the system was not leaking. (d) The vacuum was switched off and the flow module was detached from the bottom of the Petri dish. While still submerged, the flow module was positioned above the glass slide, and aligned with the endothelial cell populations using the alignment tool. (e) The flow module was carefully pressed in place and vacuum applied to seal it to the glass slide. (f) The flow module and attached endothelial slide was removed from the alignment tool in the Petri dish and dried before transfer to a microscope for imaging.