BMC Cancer 2009, 9:398 http://www.biomedcentral.com/1471-2407/9/398 stimulated and the non stimulated whole blood cells after etoposide treatments. The analysis was performed with a fluorescence microscope (Olympus BX60); 4 fields and at least 400 cells per slide were scored. The results were expressed as percentage of cells alive relative to controls. Single cell gel electrophoresis The alkaline comet assay was performed essentially as described previously [33]. Briefly, after the experimental treatment was applied, 20-μl samples of whole blood, both PHA-stimulated and non-stimulated, were dissolved in 0.5% LMPA, spread onto microscope slides precoated with 0.5% agarose, and covered with an additional 0.5% LMPA layer. The cells were then lysed in a high salt and detergent solution (2.5 M NaCl, 10 mM EDTA, 10 mM Tris pH 10, with fresh 10% DMSO and 1%Triton x-100), for at least 1 h at 4°C. Subsequently, the cells were placed in a horizontal electrophoresis chamber and exposed to an alkaline solution (300 mM NaOH, 1 mM Na2EDTA, pH >13) for 20 min to allow the DNA to unwind. For DNA electrophoresis, a 25-V electric current (300 mA, 0.8 V/cm) was applied for 20 min. All technical steps were conducted under very dim indirect light. After electro- phoresis, the slides were gently removed and the alkaline pH was neutralized by application of 0.4 M Tris, pH 7.5. The slides were dehydrated in two steps with absolute eth- anol for 5 min each. Ethidium bromide (75 μl of a 20 μg/ ml solution) was added to each slide and a coverglass was placed on the gel. We performed the comet assay at pH 12.3 as described in our previous report [33]. The comet assay was also used in combination with Exo III as described by Gedik et al. [34]. Briefly, cells were centrifuged at 200 × g for 3 min at 4°C, dispersed in 75 μl 1% low melting point agarose at 37°C, placed on a microscope slide an processed for the comet assay. For each sample, we divided the gel in two parts, the upper part of the gel was incubated with ExoIII and buffer and lower part with buffer alone. The parts were separated by a coverglass. The measures of AP sites were obtained by subtraction of the mean comet assay score with enzyme buffer alone from that with ExoIII. DNA migration was analyzed on an Olympus BMX60 microscope with epifluorescence equip- ment (with a 515-560-nm excitation filter and a 590-nm barrier filter). DNA migration measurements (tail image length, in microns) were made with a scaled ocular. To identify the tail, the head of the comet was defined as the most brilliant circular region in the image. One hundred cells were scored for each treatment condition. All experi- ments were conducted in triplicate and scored in a double blind manner. Statistical analysis All statistical analyses were performed with STATISTICA software version 5 from STAT Soft Inc. USA (1996). The Mann-Whitney U test was used to determine DNA dam- age statistical differences between control cells and those treated with etoposide. Student's t test was used to com- pare cell viability, and Exo III recognition sites between control and etoposide-treated cells. For the evaluation of ALS, we used the Shapiro-Wilk "W" test which is used to probe normality [35]. We compared each treatment with its particular control (2 h non-stimulated, 2 h stimulated, 24 h non-stimulated and 24 h stimulated) and with each etoposide concentration (0.0, 2.07, 20.7, 207.0). The results reported show that W test was not significant in all case (2 h non-stimulated, W = 0.867, P = 0.06148; 24 h non-stimulated, W = 0.9093, P = 0.20902); 2 h stimu- lated, W = 0.91198, P = 0.22618; 24 h stimulated, W = 0.93917, P = 0.48735, then the hypothesis that the respec- tive distribution is normal was accepted. We performed a one way ANOVA test in order to detect if there was an effect of the etoposide concentration (0.0, 2.07, 20.7, 207.0) on the formation of akali-labile sites (ALS) in stim- ulated and non-stimulated cells at 2 h and 24 h. Thereafter, a multiple comparison test (Tukey) was used to determine the significant differences between group means, particularly we analyzed if the control differed sig- nificantly with each etoposide concentration [35]. The relationship between ALS index and net enzyme recogni- tion sites was analyzed by Pearson's correlation. Results Viability Our observations of the cells following the dual cell-stain method for PHA-stimulated and non-stimulated whole blood cells revealed that cell viability was high after etoposide treatment for 2 h and 24 h. As shown in table 1, we observed cell viability rates that exceeded 70%. Single cell gel electrophoresis SCGE at pH >13, which reveals SSBs, DRSs and ALSs, revealed a dose-dependent effect of etoposide treatment (2.07, 20.7 and 207 μM) on cells treated for 2 h and 24 h (Figure 1A, B respectively). We observed an increase in DNA-damage in all etoposide treatments showing the highest effect at 207 μM with respect to the control. How- ever, SCGE assay performed at pH 12.3, which detect SSBs and DRSs, but not ALSs, showed genotoxic effects in the PHA-stimulated whole blood cells only at both treatment durations (Figure 2). These results demonstrate oxidative DNA damage generated by etoposide in non-stimulated blood cells, among others sources of ALS generation [35]. 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