Aging Aging

146 Clingen, Lowe, and Greenmore readily to in vitro studies of aging, and to studies of genotypes that mightinfluence the aging process.In addition to interindividual variation, intraexperimental variation shouldbe considered. It is essential to repeat experiments several times, to confirmconsistency of effects. A minimum of duplicate slides in three independentexperiments is recommended. This is particularly important in monitoringpopulations for evidence of preexisting or endogenous DNA damage. Age-relateddifferences may be small and experiments may not have built-in controls fordamage arising from poor experimental technique. As a guide to what can beachieved within a given study, one worker can readily perform and score oneexperiment of eight sets of duplicate slides per day.2. Materials2.1. Cells and TissuesWe will describe the use of the assay with (1) freshly isolated human mononuclearcells, and (2) cultured normal human fibroblasts2.2. Slides, Agarose, and Chemicals1. Slides. Fully frosted (on one surface) glass microscope slides 76 × 26 mm(Chance Propper, Smethwick, West Midlands, UK) (see Note 1). Coverslips,22 × 22 mm, thickness no. 1.5 (Merck, Leicester, UK).2. Agarose. Sigma type I (Sigma, Poole, Dorset, UK); NuSieve GTG low meltingpoint agarose (FMC BioProducts, Rockland, ME, USA).3. Culture medium, as appropriate for each cell type. We incubate freshly isolatedmononuclear cells in RPMI 1640 with phenol red (Life Sciences, Paisley,UK), supplemented with 10% pooled human AB serum, 200 IU/mL ofPenicillin, 200 µg/mL of streptomycin, 2 mM L-glutamine (Gibco-BRL, Paisley,UK), and 200 µg/mL of sodium pyruvate (Sigma, Poole, Dorset, UK). Forfibroblasts, we use minimum essential medium (MEM) containing 15% fetalcalf serum (FCS), 100 IU/mL of penicillin, 100 µg/mL of streptomycin, and2mM L-glutamic acid. Histopaque is from Sigma (Poole, Dorset, UK), humanAB serum pools from Colindale Blood Transfusion Centre (London, UK) andFCS from PAA Laboratories GmbH (Linz, Austria). Where indicated, RPMI1640 without phenol red is used.4. Dulbecco’s “A” phosphate-buffered saline (PBS, tablets from Oxoid,Basingstoke, UK). Bacto trypsin is from Difco Laboratories (Detroit, MI, USA).The contents of one vial are dissolved in 200 mL of PBS. Trypsin-EDTA contains40% v/v trypsin solution and 0.4% EDTA in PBS. Other chemicals are fromSigma (Poole, Dorset, UK).5. All solutions are made up in double-deionized water (ddH 2 O).