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Development of a Semi-automated Instrument to Measure the Cellular Reactive Oxygen Species (ROS) Activity of Ambient Particulate Matter
SUDHEER SALANA, Yixiang Wang, Joseph V Puthussery, Haoran Yu, Vishal Verma, University of Illinois at Urbana-Champaign
Abstract Number: 234
Working Group: Health-Related Aerosols
Abstract
Several automated instruments exist today to measure the acellular oxidative potential of ambient particulate matter (PM). Moreover, a few online instruments have also been developed for the real time measurements of the acellular reactive oxygen species (ROS) activity of the ambient particles. However, no such automated system exists for measuring the cellular ROS activity, which severely limits the comparison between two types of assays. Cellular assays provide a much better assessment of ROS activity as they incorporate the biological processes involved in the PM-induced ROS generation. Considering this need, we developed a first of its kind semi-automated instrument to conduct the macrophage ROS assay using rat alveolar cells (NR8383), which is a well-established and widely used cell line for the cellular oxidative potential measurements. The instrument is capable of analyzing a batch of six samples (including one negative and one positive control) in five hours and is equipped to operate continuously for 24-hours with minimal manual intervention after every batch of analysis, i.e., after every five hours. The instrument has a high analytical precision (CoV <20% for positive controls t-BOOH, Cu (II) and ambient PM sample). The results obtained from the instrument were in good agreement with manual measurements using t-BOOH (slope = 0.83 for automated vs. manual, R2 = 0.99) and ambient samples (slope = 0.83, R2 = 0.71). We further demonstrated the ability of this instrument to analyze a large number of both ambient and laboratory samples, and developed a dataset on the intrinsic cellular ROS activity of several compounds, such as metals, quinones, PAHs and inorganic salts, commonly known to be present in ambient PM. This dataset will help in apportioning the contribution of key chemical species in the overall cellular ROS activity of the ambient PM.