Measurement of endosomal kinetics and pH. (A) Dictyostelium cells were allowed to internalize for 20 minutes a mixture of Oregon green- and Alexa 647-dextran. The cells were then washed and further incubated in medium for the indicated time. At each time point, the cellular fluorescence was determined by flow cytometry. (B) To extrapolate endosomal pH values, the Oregon green/Alexa 647 ratio was calculated at each time point. A calibration curve was obtained in each individual experiment as described in Figure 1 (inset). These results indicate that early endocytic compartments (lysosomes) are very acidic, and that endocytosed fluid phase is then gradually transferred to less acidic compartments (post-lysosomes).