Fig. 6.

ROS formation in WI-38 cultures exposed to different oxygen tensions for 24 h. The cells were washed once with phenyl red, serum-free MEM and then treated with 100 μl of DCFH-DA containing medium. The plates were incubated at 37°C for 1 h. Following incubation, the cells were washed once with the wash buffer and read (ex 494, em 518) with a Victor 2 Plate Reader (Perkin Elmer) to determine the amount of oxidized DCF in the cells. Filters used were ex 485 (Perkin Elmer Filter 11440022) and em 535 (Perkin Elmer Filter 11440023). Protein in the samples was determined using Bradford protein assay reagent (Biorad). The reagent was diluted 1:5 with ultrapure water and filtered prior to treating samples. The Victor 2 plate reader was used to determine absorbance at 595 nm. Each point is an average of eight determinations