FIG. 1.
baf raises the pH of early and late endosomes in HeLa cells. (A) HeLa cells were preincubated for 30 min at 37°C without or with 20 nM or 200 nM baf. They were then incubated for 5 min in medium containing 6 mg of FITC-dextran and 1 mg of Cy5-dextran per ml and subsequently chased in dextran-free medium in the absence or presence of baf. Cells were immediately cooled, washed with PBS, and analyzed by flow cytometry. (B) HeLa cells were continuously labeled with 6 mg of FITC-dextran and 1 mg Cy5-dextran per ml for 25 min. Cells were then further incubated in fresh medium without or with 20 nM or 200 nM baf (arrow). The average pH of labeled endocytic compartments was calculated with a pH calibration curve (see Materials and Methods). Times shown in both panels represent total time from the addition of FITC-dextran and Cy5-dextran. Values shown are means and standard deviations from two experiments (each sample was analyzed eight times).