Aging Aging

NK Cell Function in Aging 3199mL of Pico-Fluor 40 scintillation cocktail. The scintillation counting is set toobtain a counting error lower than 5%.4. Notes1. The use of normal mouse serum is also recommended to reduce nonspecific bindingwhen an indirect immunofluorescence reaction is combined with a directimmunofluorescence reaction on the same cell sample.2. To study the expression of differentiation markers on NK cells the use of doublefluorescence, using PE labeled anti-CD56 or anti CD16 MAbs, is recommended.3. Cytofluorimeters are instruments that measure cell size and phenotype by thepresence of bound fluorochrome-labeled antibodies and are widely used foranalysis of cells labeled as described earlier. A single-cell suspension labeledwith FITC- or PE-conjugated antibodies is forced through the nozzle of themachine under pressure. The light scattered and reflected by the cells and fluorescenceemitted by excited fluorochromes bound to the cell membrane aredetected, analyzed, processed, and stored by a computer. Forward light scatter or“forward scatter” (FS) measures cell size while perpendicular light scatter or “sidescatter” (SS) indicates heterogeneity of the cell structure (“granularity or complexity”).4. Most NK cells show large granular lymphocyte morphology and therefore produceforward and side scatter signals higher than other lymphocyte subsets. Thuswe do not have to use a too-restricted lymphocyte gate. This point should beborne in mind when setting gates for analyzing NK cells in whole PBMCs.5. In the lysis assays the target cells should be collected in the logarithmic phase ofgrowing, as 51 Cr labeling is poorer when quiescent cells are used.6. The optimal labeling is obtained when 51 Cr is added to a target cell pellet as driedas possible to avoid dilution by culture medium.7. One cpm per target cell can be considered an adequate total labeling. The spontaneousrelease should be as low as possible, but is considered optimal if it is lessthan 10% of the maximum release although about 30% can also be exceptionallyaccepted.8. Assaying the lytic activity of peripheral blood lymphocytes with the standard testmay present some limitations when it is possible to collect only minimal bloodsamples, as it occurs in elderly people. In these situations, the possibility of usinga low number of effector cells would greatly facilitate the performance of cytotoxicityassays. In this microcytotoxicity assay, use a 10-fold lower number ofcytolytic effector and target cells with a maintained E/T ratio (3).AcknowledgementsR. S. and C. A. are supported by grants from Spanish Ministry of Health (FIS95/1242, FIS 98/1052) and Junta de Andalucía (Spain). E. M. is supported by grantsfrom MURST (60% fund) and University of Bologna. This work was carried outunder the aegis of the European Union Concerted Action on the Molecular Biologyof Immunosenescence (EUCAMBIS; Biomed I contract CT94–1209).