EpiFlow: multidimensional single-cell epigenetic profiling by spectral flow cytometry

Ruiz-Iglesias, J.; Bovolenta, E. R.; Canizares-Moscato, L.; Isoler-Alcaraz, J.; Martin-Rodriguez, L.; Segura, J.; Enriquez-Zarralanga, V.; de Rus-Moreno, A.; Contreras-Perez, A.; Gomez-Moya, A.; Garcia-Mateos, D.; Tercero, J. A.; Desvoyes, B.; Martinez del Val, A.; Camafeita, E.; Vazquez, J.; Jimenez-Carretero, D.; Raposo Ponce, B.; Ledesma, M. D.; Reglero-Real, N.; Perea, C.; Lecona, E.; Gomez, M.; Martinez-Martin, N.; Palomer, E.

doi:10.64898/2026.03.16.711606

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bioRxiv · 10.64898/2026.03.16.711606

EpiFlow: multidimensional single-cell epigenetic profiling by spectral flow cytometry

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Abstract

The epigenetic landscape of individual cells determines their identity and function, yet current methods for profiling chromatin modifications at single-cell resolution remain low-throughput, costly, or limited in parametric depth. Here we present EpiFlow, a spectral flow cytometry-based platform that enables the simultaneous quantification of 16 epigenetic markers, including histone post-translational modifications, DNA methylation, and hydroxymethylation, at the single-cell level. We demonstrate that EpiFlow is robust across species from yeast to mammals and resolves biologically meaningful epigenetic transitions during the cell cycle, stem cell differentiation, germinal centre B cell maturation, diabetic liver remodelling, and seizure-induced chromatin reprogramming. High-dimensional integration of EpiFlow data enables cell-type classification based solely on epigenetic profiles in liver, brain, blood, and cancer. Furthermore, EpiFlow detects on-target and off-target/indirect effects of epigenetic drugs in a high-throughput-compatible format. Collectively, these results establish EpiFlow as a broadly applicable platform for single-cell epigenetic analysis in basic, pharmaceutical, and translational research.

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Ruiz-Iglesias, J., Bovolenta, E. R., Canizares-Moscato, L., Isoler-Alcaraz, J., Martin-Rodriguez, L., Segura, J., Enriquez-Zarralanga, V., de Rus-Moreno, A., Contreras-Perez, A., Gomez-Moya, A., Garcia-Mateos, D., Tercero, J. A., Desvoyes, B., Martinez del Val, A., Camafeita, E., Vazquez, J., Jimenez-Carretero, D., Raposo Ponce, B., Ledesma, M. D., Reglero-Real, N., Perea, C., Lecona, E., Gomez, M., Martinez-Martin, N., Palomer, E.. 2026-03-18. EpiFlow: multidimensional single-cell epigenetic profiling by spectral flow cytometry. https://doi.org/10.64898/2026.03.16.711606

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