Click on image to view larger version.
Figure 3. (a) Intracellular oxidative stress examined by immunofluorescence. Endothelial cells preincubated with 2',7'-dichlorofluorescein diacetate (DCFH-DA; 10 µM) for 30 min were either mock treated, treated with recombinant tumour necrosis factor (TNF)
or 100 µM H2O2 alone or in combination with polyethylene glycol-tagged superoxide dismutase (PEG-SOD) and polyethylene glycol-tagged catalase (PEG-CAT). The cells were examined 30 min after treatment at 200x magnification under a green channel (at excitation 488 nm and emission 570 nm) using a Nikon TE-2000U epifluorescence microscope. Brightfield images, showing the total number of cells present in the same field, were obtained under phase contrast at 200x magnification. (b) Histogram showing the levels of TNF-induced intracellular reactive oxygen species (ROS). Cells preincubated with DCFH-DA (10 µM) for 30 min were (i) mock treated, (ii) treated with 0.1 ng ml–1, 1 ng ml–1 and 10 ng ml–1 TNF, (iii) treated with 100 µM H2O2 or (iv) exposed to 2 Gy. The fluorescent intensity was measured 30 min after treatment or exposure by flow cytometry. The percentage of cells positive for fluorescein was calculated from the total number of cells gated. The data presented are means ± standard deviation (SD) of three independent experiments. The p-values were calculated using Student's t-test.
