Aging Aging

312 Mariani, Alonso, and Solanaresting NK cells, are frequently used for testing the antibody-dependent cellcytotoxicity (ADCC, Fc receptor-mediated lysis by NK cells).The results on the different phenotypic and functional analysis of NK cellshave demonstrated age-associated changes both in the number and function ofNK cells. Thus, although there is a general consensus that NK cytotoxicity ofperipheral blood lymphocytes is well preserved not only in healthy elderlypeople but also in centenarians, a significant expansion in the number of NKcells is also found in healthy aging (1). This indicates that NK cell killing isimpaired when it is considered on a per cell basis, as it is shown when singlecellcytotoxic assays are performed (2,3). The ability of interleukin-2 (IL-2)-activated NK cells to lyse the normally NK-resistant Daudi cell line is alsosignificantly decreased in the elderly, when compared to young people (4).However, ADCC- and LAK-mediated killing remain comparable betweenyoung and elderly subjects. The decreased NK cytotoxic capacity found in theelderly is associated with defective signal transduction (5). On the other hand,perforins are distributed in the cytoplasm of almost all the NK cells from theelderly, as in NK cells from the young, the ability to utilize perforin in thegeneration of cytolytic activity against tumor target cells is maintained in NKcells from the elderly (6).Recently Ogata et al. (7) showed that low NK cell function relates to thedevelopment of severe infections in elderly subjects and that well preservedNK cell cytotoxicity is relevant for survival. These findings strengthen theinterest in studying NK cytotoxicity in aging.In the following sections we introduce the techniques and experimentalprotocols usually employed for the analysis of NK cell phenotype and functionin the elderly. The standard immunofluorescence and cytotoxicity assayas well as the techniques for determining NK signal transduction arepresented.2. Materials2.1. Analysis of NK Cell Phenotype and Perforin Contentby Immunofluorescence1. Lymphocyte separation medium (LSM), density 1.077 g/mL (e.g., Histopaque-1077, Sigma, St. Louis, MO, USA).2. Culture medium: RPMI 1640 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES) buffer supplemented with 10% fetal calf serum (FCS),antibiotics (penicillin: 100 U/mL plus streptomycin 100 µg/mL or alternativelygentamicin 200 µg/mL) and 2–4 mM L-glutamine (all reagents from Gibco Life,Paisley, UK).3. Phosphate-buffered saline (PBS) (Gibco Life).4. Trypan blue solution: 0.2% w/v in PBS/3 mM sodium azide (Sigma).