Some of the earliest apoptotic changes occur at the cell surface. The early recognition of apoptotic cells by phagocytic cells, the significant loss of water leading to cell shrinkage, and the maintenance  of intact cell membranes, despite the cell surface blebbing observed in many cell types, indicate  significant changes have occurred in the plasma membrane. One of the better understood cell surface  modifications is exposure of phosphatidylserine (PS). Normally, PS is confined to the inner layer of  the lipid bilayer. After induction of apoptosis  the PS is flipped from the inner to the outer bilayer, rendering the molecule available for detection. Annexin V is a blood clotting factor that exhibits a high calcium-dependent specificity for PS binding.  The coupling of annexin to fluorescein, or biotin, generates a direct, rapid, and simple method for the detection of  apoptosis on unfixed cells.