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. 2021 Jan 18;11:1662. doi: 10.1038/s41598-021-81217-4

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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R-integrated pixel intensity (PIXI) analysis from brightfield images integrated in the PBMC XF assay workflow. (a) Method workflow for PBMC XF assay data normalization using brightfield image analysis. (b) Original pre-XF assay brightfield images (top, 4 × objective) were processed using an in-house generated R script, generating images in which the total pixel intensity was quantified (bottom). A magnification from the middle of the brightfield image is shown for each seeding density (n = 14–16), for one donor. (c) Effect of 0%, 10% and 25% image border cropping on the size of the processed image at a seeding density of 200,000 cells/well. Images have the same scale bar. (d) Effect of border cropping percentage (0–25%) on the curve fit of plated cells versus total pixel intensity values.