20 well cometslides

DNA strand breaks were determined on frozen BAL cells suspension, lung (3×3mm of left lobe) and liver tissue (3×3mm piece of the median lobe). Sample preparation and comet analysis has previously been described in detail (36 (link)). Briefly, BAL cells, lung or liver cell suspensions, were embedded in agarose (0.7% final concentration) on Trevigen 20-Well CometSlides™. Slides were quickly immersed into lysing solution at 4°C and stored overnight. On the next day, samples were alkaline treated and subjected to alkaline electrophoresis in ice-cold circulating electrophoresis solution (25min, 1.15V/cm, pH ˃13). Samples were neutralised, fixed and later stained by SYBRGreen®. Comets were scored by the fully automated PathFinder™ system (IMSTAR, France). DNA strand breaks were quantified as %DNA in the comet tail (%TDNA) and the comet tail length (TL). The day-to-day variation and electrophoresis efficiency was controlled by including phosphate-buffered saline exposed and 60 µM hydrogen peroxide-exposed A549 cells as negative and positive controls, respectively, on all comet slides. Control cells were exposed for 30min at 4°C as described (36 (link)). The day-to-day variation for the comet assay analysis on BAL, lung and liver tissue (n = 19 for each tissue) were 19.3%, 13.1% and 16.7%, for TL, respectively.

DNA strand breaks were determined on frozen BAL cells suspension, lung (3 × 3 mm of left lobe) and liver tissue (2 × 2 mm piece of median lobe). Organ samples were snap frozen directly after dissection and kept at −80°C until analysis. Sample preparation and analysis was previously described in detail [Jackson et al., 2013 (link)]. Briefly, BAL cells, lung, or liver cell suspensions were embedded in agarose (0.7% final concentration) on TREVIGEN 20-Well CometSlides™. Slides were quickly immersed into lysing solution at 4°C and stored overnight. The next day, samples were alkaline treated and subjected to alkaline electrophoresis (pH >13) in ice cold circulating electrophoresis solution. Samples were neutralized, fixed, and later stained by SYBRGreen®. Comets were scored by the fully automated PathFinder™ system (IMSTAR, France). DNA strand breaks were quantified as % DNA in the comet tail (%TDNA) and the comet tail length (TL). The day-to-day variation and electrophoresis efficiency was validated by including PBS exposed and 60 µM H₂O₂-exposed A549 cells as negative and positive controls, respectively, on each slide. Control cells were exposed for 30 min at 4°C as described [Jackson et al., 2013 (link)]. The day to day variation including all slides (n = 21) from this experiment was 28%.