Microfluidic single-cell measurements of oxidative stress as a function of cell cycle position.

Single-cell measurements routinely demonstrate high levels of variation between cells, but fewer studies provide insight into the analytical and biological sources of this variation. This is particularly true of chemical cytometry, in which individual cells are lysed and their contents separated, compared to more established single-cell measurements of the genome and transcriptome. To characterize population-level variation and its sources, we analyzed oxidative stress levels in 1278 individual Dictyostelium discoideum cells as a function of exogenous stress level and cell cycle position. Cells were exposed to varying levels of oxidative stress via singlet oxygen generation using the photosensitizer Rose Bengal. Single-cell data reproduced the dose-response observed in ensemble measurements by CE-LIF, superimposed with high levels of heterogeneity. Through experiments and data analysis, we explored possible biological sources of this heterogeneity. No trend was observed between population variation and oxidative stress level, but cell cycle position was a major contributor to heterogeneity in oxidative stress. Cells synchronized to the same stage of cell division were less heterogeneous than unsynchronized cells (RSD of 37-51% vs 93%), and mitotic cells had higher levels of reactive oxygen species than interphase cells. While past research has proposed changes in cell size during the cell cycle as a source of biological noise, the measurements presented here use an internal standard to normalize for effects of cell volume, suggesting a more complex contribution of cell cycle to heterogeneity of oxidative stress.
(© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)