Aging Aging

Comet Assay of DNA Damage 151anode (we use two rows of eight slides). Slides should be placed close togetherand all spaces filled with blanks to prevent any movement.5. A volume of electrophoresis buffer at the correct temperature (see Note 16) isgently added to just cover the slides (see Note 17). The slides are incubated for afixed period for unwinding (we use typically 40 min; see Note 16).6. Electrophoresis. An electric current is applied (we use 20 V/24 min).7. Remove some electrophoresis buffer by suction. Gently remove the slides andplace them on a staining tray. Gently rinse with neutralizing buffer, stand for5 min, drain, and repeat twice.8. Place 35 µL of ethidium bromide solution on the surface of the agarose of eachslide and add a coverslip.9. Score slides as soon as possible. For storage, place slides at 4°C in the dark overmoist tissue in a closed box (see Note 18).3.5. Comet Assay for Detecting Endogenous Levelsof Oxidative DNA DamageFreshly isolated mononuclear cells exhibit little or no single strand breakagein the standard Comet assay as described in Subheading 3.4. However, lowsteady-state levels of oxidized purines and pyrimidine bases can be detectedusing Endo III and FPG repair enzymes.A typical assay will consist of samples from four donors. Each sample mustbe incubated both with and without enzyme, giving two sets of duplicate slidesfor each donor.1. Slides are prepared as outlined in Subheading 3.2. and lysed immediately.2. After lysis the slides are drained by carefully blotting their sides on tissue andwashed by placing in a fresh staining trough containing 150 mL of enzyme bufferat 4°C for 5 min. Replace the buffer and repeat twice.3. Slides are drained and placed on a tray. Add 50 µL of diluted enzyme (see Note 19)to the surface of the agarose and add a coverslip to spread the enzyme solutionuniformly. Each set of duplicate slides treated with enzyme must have as a control,duplicate slides treated with buffer minus enzyme. Incubate for 1 h at 37°C in adark humidified atmosphere.4. Place the slides in the gel box and add electrophoresis buffer to stop enzymeactivity. For detection of enzyme-sensitive sites, we normally omit the unwindingstep and apply electrophoresis immediately. However, for detecting low levelsof endogenous oxidative damage it may be advantageous to retain anunwinding step to increase the sensitivity of the assay.5. Electrophoresis and subsequent steps are as described in Subheading 3.4.6.3.6. Repair of Oxidative Base DamageThe rate of repair of oxidative base damage can also be determined usingeither Endo III or FPG. In this protocol sufficient controls are required to takeinto account levels of endogenous damage (untreated cells with enzyme) and