Fig. 2

Prima-1 and 3-BrPA cooperate to increase ROS. ROS intracellular generation was assayed in octuplicates in adherent A549 cells seeded in 96 well plates 10 h after exposure to the indicated treatments in medium supplemented with 5 mM glucose, 2 mM glutamine and 5% dialyzed serum. This was quantitated using DCFH-DA (Life Technologies), a cell permeable non-fluorescent compound that can be hydrolyzed by intracellular esterases to DCFH, which fluoresces green when oxidized by H₂O₂. Cells were exposed to 20 μM DCFH-DA together with 20 μM LavaCell (Active Motif. Carlsbad, California 92008, USA) for 30 min. The latter is also a cell-permeable, non-toxic compound that stains membranes of live cells providing an orange-red emission (560–580 nM). Cell-associated fluorescence was determined using the signal thresholding algorithms identify fluorescence above the solution background from which fluorescent cells are identified for calculation of morphological and fluorescent parameters in an Isocyte argon laser spectrofluorometer identifying channel 1 green fluorescence (510–540) normalized to channel 3 orange-red cell fluorescence (560–580 nm). *denotes significance between treated cells relative to controls