Monitoring volume changes in red cell ghosts by means of a trapped fluorescent probe

A method is described for monitoring changes in the volume of red cell ghosts by means of a fluorescent probe trapped inside them.. The fluorophore, 8-hydroxy-1,3,6-pyrenetrisulfonate, is partially quenched by the residual hemoglobin in the ghosts. When the ghosts swell, the hemoglobin concentration is reduced, the quenching is somewhat relieved and the fluorescence output increases. Opposite changes occur when the ghosts shrink. Fluorescence intensity is linearly related to ghost volume for both swelling and shrinking, but there is a larger change in fluorescence for shrinking from the isotonic volume than for an equivalent swelling. This method has been used to follow the swelling phase of dye-loaded ghosts suspended in a solution of a penetrating nonelectrolyte in isotonic saline.