Non-linear relationship between fluorescence and membrane potential

The fluorescence intensity of the cynanine dye DiO-C₆-(3)_in 0.32% suspension of Ehrlich ascites tumor cells was determined under various potassium concentrations. In addition, the fluorescence levels of cell-free buffer solutions and those of supernatants were measured. For every potassium concentration the partition of the dye between cells and medium was calculated and its relation to the potassium gradient was given. A model was developed which assumes the fluorescence of a cell suspension to be the sum of the fluorescence signal of dye in the extracellular medium and that of cell-associated dye. A calibration curve of fluorescence vs. membrane potential was constructed. Neither the fluorescence of the cell suspension nor that of the supernatants was a linear function of the membrane potential. The limitations of membrane potential determination by fluorimetric methods are discussed.