A NOVEL FLOW CYTOMETRIC METHOD FOR QUANTIFYING PHAGOCYTOSIS OF APOPTOTIC CELLS

Many eukaryotic cell types are capable of specific recognition and pha gocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these m echanisms, we designed a novel fluorescence-based method to quantify p hagocytosis in vitro using endothelial cell engulfment of apoptotic ce lls as a model, The B-cell Line WEHI-231 was labeled with the fluoroph ore 6)-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA) and the n induced to undergo apoptosis by crosslinking cell surface immunoglob ulin. An endothelial cell Line was subsequently allowed to ingest thes e TAMRA-labeled apoptotic lymphocytes. After 24 h, nonbound lymphocyte s were removed and the monolayers were dissociated, Any nonphagocytose d lymphocytes that remained tightly bound to the endothelial cells wer e then indirectly immunofluorescein labeled for the pan leukocyte-spec ific marker CD45, Flow cytometric analysis of the cells distinguished three endothelial cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA(+)CD45(+)); 2) endothelial cells containing p hagocytosed apoptotic lymphocytes (TAMRA(+)CD45(-)); and 3) endothelia l cells that were not associated with lymphocytes, The identification of these populations was verified by confocal microscopy of sorted cel ls. The method described herein will facilitate detailed studies on ph agocytic recognition of apoptotic cells and should have broad applicat ions to other phagocytic cell systems. (C) 1997 Wiley-Liss, Inc.