Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis.

Chlorambucil-induced apoptosis was assessed by three different flow cytometric methods in B-cell chronic lymphocytic leukaemia (B-CLL) cells cultured in vitro and the results were compared with those derived from the morphological assessment of the same samples. Spontaneous apoptosis was consistently observed in the control cultures in the absence of drug but this accounted for less than 12% of all cells in every case. The methods under investigation were the Annexin V labelling assay, the terminal deoxynucleotidyl transferase (TdT) end-labelling assay and the labelling of a 38 kDa mitochondrial membrane protein (7A6 antigen) which is exposed on cells undergoing apoptotic cell death (Apo2.7 assay). The Annexin V assay consistently stained a higher percentage of cells and with a greater separation between the positive and negative cell populations. We conclude that the phosphatidyl serine translocation to the outer leaflet of the cell membrane following an apoptotic signal, as labelled by Annexin V, probably occurs before the development of the DNA strand breaks or the exposure of 7A6 antigen in those cells triggered to die by apoptosis.