TockyLocus: quantitative analysis of flow cytometric fluorescent timer data in Nr4a3-Tocky and Foxp3-Tocky mice
File(s)
Published version
Author(s)
Ono, Masahiro
Type
Journal Article
Abstract
Fluorescent Timer proteins undergo a time-dependent shift from blue to red fluorescence after translation, providing a temporal record of transcriptional activity in Timer reporter systems. While Timer proteins are well suited for studying dynamic cellular processes such as T cell activation using the Timer of-Cell-Kinetics-and-Activity (Tocky) framework, quantitative analysis of Timer-based flow cytometry data has yet to be fully standardised. In this study, we optimise quantitative analysis methods for the key parameter within the Tocky framework, Timer Angle, and introduce TockyLocus, an open-source R package that implements a five category scheme based on biologically grounded angular intervals (designated as Tocky Loci). This approach is validated using both simulated and experimental datasets and enables downstream statistical testing and visualisation of transcriptional dynamics in flow cytometry data. Using computational modelling of Timer protein kinetics, we define transcriptional dynamics in relation to key anchoring points in Timer Angle values at 0◦, 45◦, and 90◦. Comprehensive simulations with synthetic spike-in datasets further demonstrate the robustness of the five-locus approach, which captures the three key points and the intermediate regions between these points. Building on the TockyPrep pre processing framework, we systematically evaluated categorisation schemes ranging from three to seven loci on real-world datasets from Nr4a3-Tocky and Foxp3-Tocky mice. The five-locus model emerged as optimal, showing significant advantages in balancing biological interpretability and statistical robustness. Optimised algorithms implemented in the TockyLocus package now standardise quantitative analysis of Timer Angle data, enabling reproducible interpretation without reliance on arbitrary gating or complex assumptions. In summary, the five-locus categorisation of Timer Angle data effectively links underlying biological dynamics to the percentage of cells in each Tocky Locus, providing a robust and interpretable framework for investigating transcriptional dynamics in immunology and related fields.
Date Issued
2025-09-26
Date Acceptance
2025-07-24
Citation
Biology Methods & Protocols, 2025, 10 (1)
ISSN
2396-8923
Publisher
Oxford University Press
Journal / Book Title
Biology Methods & Protocols
Volume
10
Issue
1
Copyright Statement
© The Author(s) 2025. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
License URL
Identifier
10.1093/biomethods/bpaf060
Subjects
Fluorescent Timer
Tocky
Foxp3
Nr4a3
T cell receptor signalling
flow cytometry
Publication Status
Published
Article Number
ARTN bpaf060
Date Publish Online
2025-08-26