Aging Aging

152 Clingen, Lowe, and Greenthe formation of single-stranded breaks (treated cells without enzyme). Consequently,untreated and damaged cells incubated with or without enzyme arerequired for each time point. The yield of enzyme sensitive sites (ESS) is calculatedfrom increase in Comet length as follows:ESS = [(treated cells with enzyme) – (treated cells without enzyme)] – (1)[(untreated cells with enzyme) – (untreated cells without enzyme)].A typical assay will consist of 16 slides from a single donor, duplicate controlslides incubated with and without enzyme, and three sets of duplicate slidestreated with the same concentration or dose of damaging agent and incubatedwith and without enzyme for defined times.1. Prepare slides as outlined in Subheading 3.2.2. Treat with DNA damaging agent on the slide over ice to minimize cellular repairof damage.3. To establish initial levels of damage immediately place duplicate control andtreated slides into lysis mixture.4. For repair, wash remaining slides 3× with 150 mL of medium (without damagingagent), and add 100 µL of medium and a coverslip to the agarose layer.5. Incubate and at defined time intervals place in same lysis mixture as described inSubheading 3.6.3.6. After the last slides have been placed in lysis, incubate at 4°C for a further 1 h.7. Subsequent steps are as described in Subheading 3.5.2.3.7. Measuring Rates of Overall Excision RepairThis assay makes use of the cell’s own DNA repair capacity to reveal DNAdamage. Rates of excision repair can be established by incubating cells withinhibitors of DNA resynthesis either immediately or at later times after treatmentwith a DNA damaging agent. It is particularly suitable for studies usingagents such UV-C radiation which, at the doses used in the Comet assay, do notproduce detectable levels of direct strand breaks.A typical assay will consist of 16 slides from a single donor, duplicate controlslides, and seven sets of duplicate slides treated with the same concentrationor dose of damaging agent and incubated at defined time periods for repair.1. Prepare slides as outlined in Subheading 3.2.2. Remove coverslip and UV-irradiate cells embedded in agarose (see Note 12).3. For initial rate of excision repair overlay duplicate control and treated slides with100 µL of medium containing 100 µM cytosine arabinoside and 10 mM hydroxyurea.Incubate for 1 h in a humidified atmosphere at 37°C and transfer to lysismixture at 4°C (see Note 20).4. To follow the progress of repair overlay remaining slides with 100 µL of mediumwithout cytosine arabinoside and hydroxyurea and incubate at 37°C for varioustimes up to 4 h.