Role of ROS generation in EG-porphyrin-mediated cell death.

<p>(<b>A</b>) Effect ROS scavengers on cell viability after PDT. 4T1 cells were loaded with EG-porphyrin derivatives for 16 h, washed and then pre-incubated for 1 h with L-histidine (20 mM), trolox (5 mM), NAC (15 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM) before light exposure. Similarly, HL60 cells were pre-incubated with L-histidine (20 mM), trolox (4 mM), NAC (5 mM), tiron (10 mM), DMSO (1%), PEG-catalase (300 U/ml) and sodium pyruvate (Na Pyruv) (10 mM). After irradiation the cells were further cultivated and cell viability was determined 24 h post-PDT by the trypan blue exclusion method. Cells treated with EG-porphyrin derivatives without irradiation were used as controls (dark). The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.050, ** P<0.01 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of L-histidine, trolox, NAC, tiron and DMSO. (<b>B</b>) ROS detection. 4T1 cells incubated with EG-porphyrin derivatives were washed and loaded with 10 µM APF(3′-(p-aminophenyl) fluorescein) in the presence or absence of L-histidine (20 mM) or DMSO (1%) for 30 min. Dye-loaded cells were then stimulated with UV light for 10 s under a DM IRB microscope (Leica) and fluorescence images were acquired immediately using a ×10 objective. (<b>C</b>) <i>In situ</i> detection of ROS generation. 4T1 cells grown on coverslips were sequentially loaded with porphyrin derivatives and APF probe. After 30 min incubation with the APF probe cells were exposed to UV light for 10 s and fluorescence images were acquired immediately using a ×63 objective. Fluorescent images of EG-porphyrin derivatives (left panels), APF probe (middle panels) and their overlay (right panels). Scale bars: 10 µm.