An A549 Cell-Based Approach Using Repeated Fluorescence Readouts for Assessing Reactive Oxygen Species Activity of...

Exposure to atmospheric particulate matter (PM) is a major public-health concern, in part because PM can perturb cellular redox homeostasis. This study evaluates an in vitro A549/DCFH-DA approach using repeated fluorescence readouts to assess PM2.5-induced oxidative activity. Untreated and assay-specific controls were combined with zymosan and NIST Standard Reference Material^® 2584 suspended in PBS, and fluorescence was monitored at multiple readout times over a 15 min-6 h window. Method performance was characterized using the coefficient of variation (CV) and signal-to-noise ratio (SNR). A dedicated three-concentration SRM 2584 series (0.02, 0.05 and 0.10 mg mL^-1) further showed readout-dependent concentration behaviour: at 60 min the untreated-control-corrected mean response increased across the tested concentrations and followed an approximate descriptive linear trend (R² = 0.90), whereas earlier readouts were non-monotonic. Substrate-related effects were examined using paired PTFE and quartz filters. Among the eight matched PTFE-quartz pairs included in the regression analysis, zero-intercept fits showed slopes close to unity for both mass- and air-volume-normalized responses (0.90 and 0.99, respectively; R² ≈ 0.99), demonstrating strong proportional agreement within this comparison set; the limited number of pairs does not support universal substrate interchangeability. Application to chemically characterized field PM2.5 samples from an urban-background site and a high-altitude site showed that DCFH-DA fluorescence did not track PM mass alone and is interpreted in terms of exploratory associations with particle composition, rather than causal effects of individual constituents. Taken together, these findings support the use of the method-performance-characterized workflow for assessing oxidative responses to field-collected PM2.5 across multiple readout times and for investigating their associations with particle chemical characteristics.