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. 2018 May 22;8:8003. doi: 10.1038/s41598-018-26172-3

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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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Analysis of the change in motility parameters either side of the maximum calcium signal tracked. (A) CD4⁺ T cells were perfused over sAgs-spiked or untreated MDMs. Scatter plots show individual “objects” (T cells) identified in every image frame from live imaging experiments with cells from one donor. The change in displacement measures how far that object has moved since the previous time frame. Objects with 340:380 ratios greater than two standard deviations above the mean (i.e. ratios >1.636) of maximum calcium intensities are highlighted in red. (B) Diagram of the pre:post max 340:380 ratio analysis adopted to determine differences in motility before and after calcium flux within a background of a flow rate that decreases over time and can differ between regions imaged in different lateral positions. (C) Comparison of meandering index (MI) ratio, Euclidean distance ratio, length ratio and speed ratio before and after the max 340:380 signal. Each point represents an individual cell measured from donor (n = 3) samples treated with sAgs [Mean, x; Bars: median ± interquartile range. Statistical analysis: Mann Whitney U test].