Aging Aging

Immunochemical Assay for DNA Damage 16314. 0.1 M MgCl 2 solution: Dissolve 2.03 g of MgCl 2·6H 2 O in 100 mL ofMQ-water.15. Reaction buffer: Dissolve 5.26 g of diethylamine (DEA) in 5 L of MQ-water andadd 50 mL of 0.1 M MgCl 2 , adjust pH to 9.8, and divide over ten 500-mL flasks.Can be stored for 6 mo in the cold room. Before use, raise the temperature toroom temperature. Check the pH (at room temperature) and, if needed, adjust thepH to 9.8. After use place the flask back in the refrigerator.16. 4-methylumbelliferyl phosphate (MUP) dilithium salt·3H 2 O: Boehringer, cat. no.405 663, check expiration date. Mol wt: 322.1. Store in the dark at –20°C.17. MUP stock solution: Dissolve the whole contents of a flask of MUP (approx 250 mgnet weight by back weighing) in reaction buffer, pH 9.8, in clean glassware at aconcentration of 20 mM (6.44 mg/mL), and divide into 1-mL portions in 1.5-mLEppendorf tubes (with cap) and store at –20°C. Can be stored for 6 mo (in the dark).18. MUP solution: Thaw the required amount of MUP stock solution and dilute 1:100in reaction buffer, pH 9.8. The remaining MUP stock solution can be placed backin the freezer, marked with ink pen. Prepare this solution no sooner than 2 hbefore use.19. Pipet tips (white opaque), 1–200 µL, Costar, no. 4862 (see Note 2).20. Pipet tips (blue), 100–1000 µL, Eppendorf, no. 0030 015.002.21. Pipets, P100, P200, and P1000, Gilson.22. 12-Channel pipet, 25–200 µL; Micronic (Macap) cat. no. 200-12.23. 96-Well dispenser, Transtar 96, adjustable volume, cat. no. 7605 and Transtarelevator cat. no. 7606, Costar.24. Plate washer, Skanwasher 300, Skatron.25. 12-Channel handplate washer, with tube connected to a polypropylene flaskwith PT.26. Sonicator/cell disruptor, Ultrasonics, W370 with microtip.27. Humidified incubator of 37°C.28. Fluorescence microtiterplate reader, Cytofluor II (Perseptive Biosystems,Framingham, MA, USA).29. Plate vibrators, Titertek (Flow) and IKA (model MTS4).30. Reaction tube mixers, Vibrofix, IKA, model VF1.3. Methods3.1. Preparation of DNA Samples (Somatic Cells)1. Cells (e.g., human total WBC) in 1 mL of blood are irradiated with 0, 1, 2 or 5 Gy60 Co-rays at 0°C. (Note: Blood may contain infectious particles; wear appropriateprotective devices.)2. At yellow light, at 20 + 1°C (see Note 1): 200 µL of alkaline solution of pH 12.3or 12.4 is added quickly, but avoiding shaking afterwards, to 30 µL of 10× dilutedblood (diluted in RPMI 1640 + 10% FCS, or in PBS) on the bottom in a clustertube (in 96-wells tray; see Note 3). The pH chosen depends on the extent ofunwinding that is required.