Aging Aging

160 van der Schanswithin 1 h, after doses as low as 0.2 Gy. The precoating of microtiter plateswith anti-ssDNA antibody enables the detection of ssDNA fragments directlyin alkali-treated blood samples. Prelabeling and isolation of the nucleated cellsfrom the blood is not necessary. Only a few microliters of blood is required andthe assay can be applied simultaneously on a large number of samples.The method is also applicable to other cell types that can be obtained insuspension and can be applied in a large variety of studies on induction andrepair of damage, including that on aging (3).A special application is that on sperm cells (4). Because in these cells theDNA is rather tightly packed the alkaline treatment conditions have to be modifiedto release the DNA. Owing to the absence of any repair in these cells anaccumulation of (oxidative) damage occurs, resulting in a background level ofsingle-stranded breaks corresponding to that induced by 50–200 Gy γ-rays.2. Materials2.1. Preparation of DNA Samples (Somatic Cells)1. Blood collected in evacuated glass tubes containing EDTA.2. RPMI 1640 culture medium containing 10% fetal calf serum.3. Cultured mammalian cells.4. MQ-water: Demineralized water purified further by filtration through a Milli-Qfilter (class 1 according to ISO 3696).5. 1 M NaOH solution: Dissolve 3.93 g of NaOH (in tablet form) in (by weighing)98 g of MQ-water (or proportionally other amounts) in a 100-mL medium flask.Close with two tissues of parafilm covered with a loosely tightened screwcap.Can be stored at room temperature for 2 mo.6. 1.3 M NaCl solution: Dissolve 38.0 g of NaCl in MQ-water and fill up to 500 mL.Can be stored at room temperature for 2 mo.7. Alkali solution: Add 2.00 mL of 1 M NaOH or 2.50 mL of 1 M NaOH to 103 g ofa 1.3 M NaCl solution (calculated pH 12.3 or 12.4, respectively). Prepare on theday of use.8. 0.25 M NaH 2 PO 4 solution: Dissolve 8.63 g of NaH 2 PO 4·H 2 O in MQ-water andfill up to 250 mL.9. Polystyrene cluster tubes, Costar, cat. no. 4408.10. Polystyrene tubes with cap, 12.4/75 mm, Greiner, no. 120161.11. 1.5-mL Eppendorf tubes.12. Pipet tips (white opaque), 1–200 µL, Costar, no. 4862 (see Note 2).13. Pipet tips (blue), 100–1000 µL, Eppendorf, no. 0030 015.002.14. Pipets, P100, P200 and P1000, Gilson.15. 12-Channel pipet, 25–200 µL; Micronic (Macap) cat. no. 200–12.16. Sonicator/cell disruptor, Ultrasonics, W370 with microtip.17. Humidified incubator of 37°C.18. Reaction tube mixers, Vibrofix, IKA, model VF1.