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. 2022 Dec 2;12:20817. doi: 10.1038/s41598-022-24371-7

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© The Author(s) 2022

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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Exemplary ridge plots used to threshold antibodies and to evaluate antigen expression. (A) shows an exemplary antigen (CD3) which was detectable at all dilutions. In contrast, CD244 shown in (B) was not detected at the lowest concentration (0.04 ×). (A, B) Antibody thresholds, indicated with red lines, were set to the upper limits of negative populations. (C) Some antigens, such as CD45, were detectable in all cells, as expected. Thus, negative populations did not exist. Here, antibody thresholds (red lines) were set to the lower limits of positive populations. If a considerable amount of expression values and the lower limit of the populations were negative, thresholds were not called (as for the 0.04 × concentration in the CD45 example). The exemplary antigen (CD178) shown in (D) was not detectable at any concentration. (A–D) Expression values were centered log-ratio (CLR) normalized.