Aging Aging

174 Wood et al.have a much higher content of oxidized bases (since the mitochondria are thesite of production of reactive oxygen species), and so might lead to a significantoverestimation of nuclear DNA damage. The best course, therefore, is toisolate nuclei first. This, in addition, avoids the possibility of DNA oxidationarising from cytoplasmic contamination with peroxisomes, or with free iron orcopper ions, that can catalyze the Fenton reaction. Liver cytoplasm is particularlylikely to provide an oxidative environment.3.1. Isolation of DNA1a. Isolation of nuclei from cultured cells: Suspend 20 × 10 6 cells in 3 mL of homogenizationbuffer with Triton X-100 in a 15-mL centrifuge tube, leave for 5 min onice, and centrifuge 10 min at 1200g, 4°C. Wash with 5 mL of Triton-free homogenizationbuffer, centrifuge for 5 min at 1200g, 4°C, and disperse the pellet wellin 1 mL of Triton-free buffer. Measure volume and make up to 4.7 mL with buffer.1b. Isolation of nuclei from human lymphocytes: Suspend 18 × 10 6 lymphocytes(expected yield from 30 mL of blood, isolated by standard density gradient sedimentationmethod) in 5 mL of homogenization buffer with Triton X-100 andcontinue as described in step 1a above.1c. Isolation of nuclei from tissue samples (e.g., liver): Weigh out approx 150 mg ofliver (fresh, or frozen under liquid N 2 ) as quickly as possible to avoid exposure toair. Add to 4 mL of ice-cold homogenization buffer (with Triton X-100). Transferto a glass homogenizer tube (Potter–Elvehjen) on ice, and break up the tissuewith about six strokes of the homogenizer over 1 min. Start at slow speed, turn upto full speed, and slow down before removing pestle from tube. Place homogenatein a 15-mL tube and centrifuge for 10 min at 1200g, 4°C. Discard supernatant,agitate pellet by shaking, add 5 mL of Triton-free homogenization buffer towash the pellet, and centrifuge for 5 min at 1200g, 4°C. Discard supernatant, andresuspend the pellet well in 1 mL of Triton-free homogenization buffer by shaking.Measure volume and make up to 4.7 mL with buffer.2. Add 10% sodium dodecyl sulfate (SDS) in HPLC grade H 2 O to the nuclear suspensionto give a final concentration of 0.6%. The pellet must be well dispersed atthis stage; otherwise SDS will not lyse all the nuclei. Gently invert tube severaltimes to mix. Incubate for 10 min at 37°C. Add 200 µL of RNase IIIA and 10 µL ofRNase T 1 . Gently invert 20× to mix, and with a wide-opening pipet aspirate up anddown twice. Incubate for 30 min at 37°C. Add 1 mg of proteinase K (usually about70 µL but varies from batch to batch). Mix by aspirating gently twice up and downwith a pipet. Incubate for 30 min at 37°C. Cool to room temperature.3. Transfer the reaction mix to a stoppered glass tube (i.e., resistant to chloroform).Add an equal volume of chloroform/isoamyl alcohol (24:1). Shake vigorouslyfor about 15 s to mix. Centrifuge in a glass centrifuge tube for 10 min at 2400g,room temperature, with no brake. Collect the upper aqueous layer, taking carenot to disturb the cloudy interface between the two phases. If the interface issolid then carefully pour off the top layer; however, if interface is not com-