Aging Aging

82 Carmody et al.4.2. TUNEL Assay4. If an assessment of necrosis is required in a given population this can be achievedprior to fixation by employing the PI exclusion assay as described in Subheading2.1.5. This avoids any clumping of cells that may occur if a 1% (w/v) p-formaldehydefixation buffer were added directly to samples.4.3. Detection of Reactive Oxygen Species6. Cells can be treated with apoptosis-inducing agents either before, after, or duringthe incubation period, depending on the time point at which ROS levels are to bemeasured.7. As a positive control for peroxide production, cells may be treated with 1 mMH 2 O 2 for 30–60 min (see Fig. 1B).AcknowledgmentsThis work was supported by the Foundation for Science and Technology(Fundação para a Ciência e a Tecnologia), Lisbon, Portugal, RP Ireland FightingBlindness, and the EU Biotech Programme.References1. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis, a basic biologicalphenomenon with wider implications in tissue kinetics. Br. J. Cancer 26, 239–245.2. Trump, B. F., Beresky, I. K., and Osornio-Vargas, A. R., eds. (1981) in Cell Deathin Biology and Pathology, Chapman and Hall, New York.3. Raff, M. C. (1992) Social controls on cell survival and cell death. Nature 365, 397–400.4. Gorman, A. M., McGowan, A. J., O’Neill, C., and Cotter, T. G. (1996) OxidativeStress and apoptosis in neurodegeneration. J. Neurol. Sci. 139, 45–52.5. Devaux, P. F. (1991) Static and dynamic lipid asymmetry in cell membranes. Biochemistry30, 1163–1173.6. Martin, S. J., Reutelingsperger, C. P. M., and Green, D. R. (1996) Annexin V- aspecific probe for the detection of phosphatidylserine exposure on the outer plasmamembrane leaflet during apoptosis, in Techniques in Apoptosis: A Users Guide(Cotter, T. G. and Martin S. M., eds.), Portland Press, London, pp. 107–119.7. Martin, S. J., Reutelingsperger, C. P. M., McGahon, A. J., Rader, J., van Schie, R.C. A. A., La Face, D. M., and Green, D. R. (1995) Early redistribution of plasmamembrane phosphatidylserine is a general feature of apoptosis regardless of theinitiating stimulus: Inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med.182, 1545–1556.8. Swairjo, M. A., Concha, N. O., Kaetzel, M. A., Dedman, J. R., and Seaton, B. A.(1995) Ca 2+ -bridging mechanism and phospholipid head group recognition in themembrane-binding protein Annexin V. Nat. Struct. Biol. 2, 968–974.9. Arends, M. J., Morris, R. G., and Wyllie, A. H. (1990) Apoptosis: the role of theendonuclease. Am. J. Pathol. 136, 593–608.