In the early stages of apoptosis changes occur at the cell surface, which until 
now have remained difficult to recognize. One of these plasma membrane 
alterations is the translocation of phosphatidylserine (PS) from the inner side 
of the plasma membrane to the outer layer, by which PS becomes exposed at the 
external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding 
protein with high affinity for PS. Hence this protein can be used as a sensitive 
probe for PS exposure upon the cell membrane. Translocation of PS to the 
external cell surface is not unique to apoptosis, but occurs also during cell 
necrosis. The difference between these two forms of cell death is that during 
the initial stages of apoptosis the cell membrane remains intact, while at the 
very moment that necrosis occurs the cell membrane looses its integrity and 
becomes leaky. Therefore the measurement of Annexin V binding to the cell 
surface as indicative for apoptosis has to be performed in conjunction with a 
dye exclusion test to establish integrity of the cell membrane. This paper 
describes the results of such an assay, as obtained in cultured HSB-2 cells, 
rendered apoptotic by irradiation and in human lymphocytes, following 
dexamethasone treatment. Untreated and treated cells were evaluated for 
apoptosis by light microscopy, by measuring the amount of hypo-diploid cells 
using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish 
whether or not DNA fragmentation had occurred. Annexin V binding was assessed 
using bivariate FCM, and cell staining was evaluated with fluorescein 
isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously 
with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The 
test described, discriminates intact cells (FITC-/PI-), apoptotic cells 
(FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing 
traditional tests the Annexin V assay is sensitive and easy to perform. The 
Annexin V assay offers the possibility of detecting early phases of apoptosis 
before the loss of cell membrane integrity and permits measurements of the 
kinetics of apoptotic death in relation to the cell cycle. More extensive FCM 
will allow discrimination between different cell subpopulations, that may or may 
not be involved in the apoptotic process.