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. 2023 Apr 7;6:376. doi: 10.1038/s42003-023-04766-6

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© The Author(s) 2023

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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Fig. 8 — a Representative total internal reflection fluorescence (TIRF) microscopy images with average intensity projections of the first 100 frames of the basal plasma membrane of transfected HEK293T cells expressing CD86-eGFP, CD28-eGFP and human CLEC-2-eGFP showing individual fluorescent spots visualised with lookup table cyan hot (field of view = 40 × 40 μm) (scale bar = 10 μm). b Representative image of the fluorescent spot detection following application of an automated spot detection algorithm of the entire basal membrane of the cell expressing CLEC-2-eGFP. Detected spots included in the analysis are shown in green and detected background is shown in yellow in panels 1 and 2 (scale bar = 5 μm). c Fluorescence intensity decay traces (grey) for individual spots showing photobleaching steps determined by the algorithm (green line). d Photobleaching step frequency histograms determined from all accepted spots for CD86-eGFP (red), CD28-eGFP (blue) and CLEC-2-eGFP (purple) in HEK293T cells. Significance of the distributions was measured with Epps-Singleton 2 sample test where P ≤ 0.05. * = significance compared to CD86-eGFP and # = significance compared to CD28-eGFP. Data pooled from 355 to 1008 traces (n = 3 biologically independent samples.