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Abstract The recent push toward understanding an individual cell's behavior and identifying cellular heterogeneity has created an unmet need for technologies that can probe live cells at the single‐cell level. Cells within a population are known to exhibit heterogeneous responses to environmental cues. These differences can lead to varied cellular states, behavior, and responses to therapeutics. Techniques are needed that are not only capable of processing and analyzing cellular populations at the single cell level, but also have the ability to isolate specific cell populations from a complex sample at high throughputs. The new CellMag‐Coalesce‐Attract‐Resegment Wash (CellMag‐CARWash) system combines positive magnetic selection with droplet microfluidic devices to isolate cells of interest from a mixture with >93% purity and incorporate treatments within individual droplets to observe single cell biological responses. This workflow is shown to be capable of probing the single cell extracellular vesicle (EV) secretion of MCF7 GFP cells. This article reports the first measurement of β‐Estradiol's effect on EV secretion from MCF7 cells at the single cell level. Single cell processing revealed that MCF7 GFP cells possess a heterogeneous response to β‐Estradiol stimulation with a 1.8‐fold increase relative to the control.
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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 that a more complex contribution of cell cycle in heterogeneity of oxidative stress.
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- Award ID(s):
- 2126177
- PAR ID:
- 10519977
- Publisher / Repository:
- Springer
- Date Published:
- Journal Name:
- Analytical and Bioanalytical Chemistry
- Volume:
- 415
- Issue:
- 26
- ISSN:
- 1618-2642
- Page Range / eLocation ID:
- 6481 to 6490
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/s00216-023-04924-z
