QUANTIFICATION OF APOPTOSIS IN LYMPHOCYTE SUBSETS AND EFFECT OF APOPTOSIS ON APPARENT EXPRESSION OF MEMBRANE-ANTIGENS

Annexin V binding to phosphatidylserine was evaluated by flow cytometr y to examine apoptosis in different lymphocyte subsets of peripheral b lood mononuclear cells after a 24 h in vitro culture period. We also a pplied a 2 Gy dose gamma-irradition prior to incubation to evaluate th e additional apoptogenic effect of radiation on the lymphocyte subsets , Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-posit ive and CD45RA-positive cells. Natural killer cells turned out to be m ost apoptosis-resistant, In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte sub populations remained, Backgating of the annexin V-positive cells showe d that these cells had a clearly decreased forward scatter signal, The antibody binding capacity (ABC) of lymphocyte membrane antigens was d etermined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4 -phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apopto tic cells, In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the c ultures. We conclude that the change in scatter and in ABC must be con sidered in immunophenotyping experiments on cells kept in culture for 24 h, If these changes are taken into account, percentages of subpopul ations or the numbers of cells that stain positive for the studied mar kers do not significantly change. (C) 1997 Wiley-Liss, Inc.