Aging Aging

Comet Assay of DNA Damage 1535. As required, carefully drain duplicate slides and overlay agarose with 100 µLcontaining 100 µM cytosine arabinoside and 10 mM hydroxyurea. Incubate for afurther 1 h in a humidified atmosphere at 37°C.6. After 1 h, transfer slides treated with inhibitor to the same lysis mixture asdescribed in Subheading 3.7.3.7. After the last slides have been placed in lysis mixture incubate at 4°C for afurther 1 h.8. Subsequent steps are as described in Subheading 3.4.4.3.8. Scoring1. Remove the slides from the storage box, wipe dry and allow to warm to roomtemperature to avoid condensation.2. Identify fields containing Comets under the microscope and score, either using animage analysis system, or assigning Comets to arbitrary categories (see Note 21).3.9. Interpretation and Analysis1. There is extensive discussion over the most appropriate parameter to score in theComet assay, whether tail length is adequate, or whether tail moment or a relatedfunction is more appropriate. In our experience, and in most cases, the parameterchosen makes almost no difference.2. There has also been debate on the statistical distribution of tail moment and otherparameters. It is not difficult to show that Comet parameters are not normallydistributed, but statistical analysis should be based on the consistency of repeatdeterminations and experiments, where distribution of individual Comet measurementswill be far less critical.3. Nevertheless, variation in levels of damage between Comets on the same slidemay be a useful indicator of the nature of the damaging event and it is desirable tokeep a record of this information.4. Results are typically expressed as mean Comet length (or tail moment) or increaseor decrease in this parameter over untreated controls. These are arbitrary units ofDNA damage or repair. It is possible, with caution, to calibrate the number ofstrand breaks against a known damaging agent, such as ionizing radiation5. In our view the important issues are (1) design of a study with an adequate numberof subjects or observations; (2) repetition of experiments; (3) preparation anddisaggregation of the cells; and (4) consistency in scoring procedures.4. Notes1. A procedure for using standard clear slides and precoating them with agarose isavailable (29).2. Other DNA stains can be used, for instance DAPI (17) or YOYO-1 (30). Ethidiumbromide or propidium iodide are bright and show good stability.3. Other systems include Perceptive Instruments (Little Yeldham, Essex, UK). It ispossible to obtain reasonably good quantitation without image analysis, by placingnuclei into categories according to the extent of damage (31).