Figure 3: Dietary supplement of NS-5 mixture and its main components inhibit intracellular superoxide production in LPS-stimulated HUVEC: The working solutions (10 μM) of NS-5 mixture and its components were prepared by diluting the appropriate volume of each stock solution in culture medium to give the final ethanol concentration of 0.1% (v/v). Monolayers of HUVEC were maintained in culture dishes pre-coated with collagen type IV at 37°C, 5% CO2 and 95% air in EGM supplemented with 2% fetal bovine serum (FBS), 10 ng/ml human epidermal growth factor, 1 μg/ml hydrocortisone, and 12 μg/ml bovine brain extract. Cells of third passage were used for experimentation. Cells were pretreated with NS-5 mixture or its components (set of 1-6 and 7-12) for 60 min and then set 7-12 were treated with LPS (100 ng/ml) for 30 min, followed by incubation of both sets with dihydroethidium dye (5 μM) specific for superoxide detection, and images were captured by epifluorescence microscope under constant exposure time and gain. The control cells received only 0.1% ethanol (v/v) 95% in culture medium. Data are mean fluorescence intensity of the cells (n=12-16) ± SD (standard deviation). Percentages of each treatment compared to control group (vehicle) of each treatment is above the column. Values in a column not sharing a common symbol are significantly different of various groups at set 1-6, ¶, § P < 0.02; and set 7-12, ¶, §, ‡ P < 0.01.