Figure 3.

Determination of ROS generation via detection of DCFDA by flow cytometry and fluorescent microscopy. ROS generation in LP9/TERT-1 cells following (A) 8 h or (B) 24 h exposures to glass beads (non-pathogenic control), Libby six-mix, or 10 mM H₂O₂ (positive control) as determined by flow cytometric detection of DCFDA staining. White and black arrowheads indicate log shifts following exposure to H₂O₂ (10 mM; 20 min) and Libby six-mix (75×10⁶ μm²/cm²), respectively. (C) DCFDA levels were also visualized using fluorescence microscopy. White arrowheads indicate increased DCFDA levels in Libby six-mix (75×10⁶ μm²/cm²) treated cells. In the merged image, black arrowheads indicate identical areas defined by the white arrowheads, and demonstrate that the highest levels of ROS are produced in areas were Libby six-mix contacts LP9/TERT-1 cells. Surface area (x10⁶ μm²/cm²) is represented in parentheses following mineral name. Bottom panel shows co-fluorescence of Hoechst dye (red nuclear stain) and DCFDA. Scale bar = 50 μm.