BibTex format
@article{Ono:2025,
author = {Ono, M},
journal = {Biology Methods & Protocols},
title = {TockyLocus: quantitative analysis of flow cytometric fluorescent timer data in Nr4a3-Tocky and Foxp3-Tocky mice},
year = {2025}
}
Citation
RIS format (EndNote, RefMan)
TY - JOUR
AB - 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 capturesthe 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 sevenloci on real-world datasets from Nr4a3-Tocky and Foxp3-Tocky mice. The five-locus model emerged asoptimal, showing significant advantages in balancing biological interpretability and statistical robustness.Optimised algorithms implemented in the TockyLocus package now standardise quantitative analysisof Timer Angle data, enabling reproducible interpretation without reliance on arbitrary gating or complexassumptions. In summary, the five-locus categorisation of Timer Angle data effectively links underlyingbiological dynamics to the percentage of cells in each Tocky
AU - Ono,M
PY - 2025///
SN - 2396-8923
TI - TockyLocus: quantitative analysis of flow cytometric fluorescent timer data in Nr4a3-Tocky and Foxp3-Tocky mice
T2 - Biology Methods & Protocols
ER -
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