% DNA in Comet Tail
Figure 66.3 Levels of oxidative purine damage in 11 different TCC as a function of their in vitro life span, determined using a modified alkaline comet assay. Values represent the Mean ± SEM for each assay, counting 50 cells/slide.
3. For the modified comet assay, to determine levels of oxidative purine or pyrimidine damage, the following additional steps are performed:
Slides must be equilibrated in enzyme buffer (0.04 M HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8.0) and then treated with the enzyme at 37° C in a humid, dark chamber for 45 minutes, which nicks oxidatively damaged purines, creating single strand breaks.
• EndoIII treatment:
Slides are equilibrated in enzyme buffer (0.04 M HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg.ml BSA, pH 8.0) three times for five-minute washes and then treated with the enzyme at 37°C in a humid, dark chamber for 45 minutes, in this case nicking oxidatively damaged pyrimidines, creating single strand breaks.
4. Slides treated with the lesion-specific enzymes are then incubated at 37° C in a humid dark chamber for 45 minutes.
5. T cells treated with 150 ^M hydrogen peroxide for five minutes at 4°C (to induce oxidative DNA damage) should be used as internal positive controls in the modified alkaline comet assay to verify enzyme activity.
6. Following enzyme treatment (or directly after alkaline lysis in the case of the alkaline comet assay) the slides are placed in an electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH 13) for
40 minutes to allow alkaline unwinding of the DNA, and then electrophoresed at 25 V, 300 mA, for 30 minutes.
7. Following electrophoresis, the slides are neutralized using 0.4 M Tris pH 7.5 and stained with 50 ^g/ml ethidium bromide. Stained slides can then be digitally analyzed, for example, using UV microscopy and Komet 3.0 analysis software (Kinetic Imaging, UK), counting 50 cells per slide. DNA damage results are expressed as percentage DNA in the comet tail.
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