Time-Resolved Single-Cell Assay for Measuring Intracellular Reactive Oxygen Species upon Exposure to Ambient Particulate Matter

Abstract

Health risks associated with exposure to ambient particulate matter (PM) are a major concern around the world. Adverse PM health effects have been proposed to be linked to oxidative stress through the generation of reactive oxygen species (ROS). In vitro cellular assays can provide insights into components or characteristics of PM that best account for its toxicity at a cellular level. However, most current assays report cell population averages and are mostly time endpoint measurements and thus provide no temporal information. This poses limitations on our understanding of PM health effects. In this study, we developed a microfluidic assay that can measure cellular ROS responses at the singlecell level and evaluate temporal dynamic behavior of single cells. We first established a protocol that enables culturing cells in our microfluidic platform and that can provide reproducible ROS readouts. We further examined the heterogeneous ROS responses of cell populations and tracked the dynamics of individual cellular responses upon exposure to different concentrations of PM extracts. Our results show that in an alveolar macrophage cell line, cellular ROS responses are highly heterogeneous. ROS responses from different cells can vary over an order of magnitude, and large coefficients of variation at each timepoint measurement indicate a high variability. The dynamic behavior of single-cell responses is strongly dependent on PM concentrations. Our work serves as a proof-of-principle demonstration of the capability of our microfluidic technology to study time-resolved single-cell responses upon PM exposure. We envision applying this high-resolution, high-content assay to investigate a wide array of single-cell responses (beyond ROS) upon exposure to different types of PM in the future.

Oxidized and Unsaturated: Key Organic Aerosol Traits Associated with Cellular Reactive Oxygen Species Production in the Southeastern United States

“…While this study region focuses on Atlanta across two different seasons, our work demonstrates how advanced multi-instrument chemical characterization of SOA and cellular measurements provide novel insights into the components and chemical features that drive SOA toxicity. Future studies can further leverage these strategies to advance the understanding of the health impacts of SOA and its subcomponents through collaborative research between atmospheric chemistry, toxicology, epidemiology, and biotechnology. ,, Finally, long-term measurement of SOA is very limited and SOA is not directly considered in current epidemiological studies. Given the important role of SOA in PM 2.5 in cellular ROS production, long-term, advanced measurements of OA types (including different types of SOA) over a wide range of geographical regions like the ongoing ASCENT (United States, ) and ACTRIS (Europe, ) networks are critical to advance the understanding of the health impacts of SOA.…”

“…This cell line has been used for measuring cellular ROS production upon PM exposure in a number of prior studies. 11,15,[30][31][32]36,51 PM 2.5 filters were extracted following the established protocol where the filters were submerged in RPMI-1640 media and sonicated for 30 min. 11 Note that some studies suggest that antioxidants and ligands in the lung fluid may consume or complex with PM components.…”

“…Future studies can further leverage these strategies to advance the understanding of the health impacts of SOA and its subcomponents through collaborative research between atmospheric chemistry, toxicology, epidemiology, and biotechnology. 6,51,94 Finally, long-term measurement of SOA is very limited 24 ■ AUTHOR INFORMATION…”

Linking Cell Health and Reactive Oxygen Species from Secondary Organic Aerosols Exposure

“…Alveolar macrophages are the first line of defense against environmental insult . We have used this cell line for measuring cellular ROS level upon PM exposure in a number of our prior studies. ,,,,, …”

“…41 We have used this cell line for measuring cellular ROS level upon PM exposure in a number of our prior studies. 11,14,15,40,42,43 2.4. Mitochondrial Membrane Potential and Caspase 3/7 Activity Measurements.…”