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. 2018 Jan 8;8:5. doi: 10.1038/s41598-017-18501-9

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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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Comparison of the CLIQ-BID and “cell area” methods. HUVECs were infected with P. aeruginosa for different durations; images of the nuclei were acquired before cell fixation, immunostaining and acquisition of cell area images. (a) Image sets at different time points for (i) Hoechst: cell nuclei; (ii) Bright nuclei: nuclei automatically segmented and sorted for intensities below (magenta) or above (green) a fixed threshold; (iii) Vinculin: cell area detected with a cytoplasmic vinculin staining; iv) Cell area: automated thresholding of the immunostaining allowing the calculation of the field area covered by the cells. The scale bar shown on the last nuclei image corresponds to 50 µm. (b) Plots of the percentage of nuclei with intensities above the thresholds and of the percentage of area cleared by the cells after different infection durations. Error bars represent the standard deviation (n = 8).