Discrimination of early and late apoptotic cells by NBD-phosphatidylserine-labelling and time-lapse observation of phagocytosis of apoptotic cells by macrophages

Since free apoptotic cells are not detected in normal tissues, it is generally believed that apoptotic cells are removed as soon as they appear in vivo. A fluorescent derivative of phosphatidylserine, 1-palmitoyl-2-6-(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospho-L-serine (NBD-PS) is known to be incorporated into living cells, and thereafter gradually absorbed into either fatty acid-free bovine serum albumin or fetal calf serum from the outer leaflet of the cell membrane. When thymocytes were irradiated with X-ray and cultured in the presence of NBD-PS, cells became less fluorescent as apoptosis advanced, but early apoptotic cells were still positive for NBD-PS. We then co-cultured such early apoptotic thymocytes with resident peritoneal macrophages. Upon examination under a time-lapse fluorescence microscope, it was found that the attachment of early apoptotic cells to macrophages does not cause rapid phagocytosis, as compared with late apoptotic cells, suggesting the possibility that, in contrast to the widely held view, early apoptotic cells may not be quickly removed by phagocytes in vivo.