pH calibration curves. (A) A combination of ammonium chloride and of sodium azide allows neutralization of endosomal pH. To test the effect of various treatments on lysosomal pH, cells having internalized FITC-dextran were incubated at pH = 7.4 in medium alone (Medium), or in medium containing 10 μM nigericin (Nigericin), 40 mM ammonium chloride (NH
Cl), or 0.1% sodium azide (NaN
), as indicated. FITC fluorescence was then measured in a Fluorescence Activated Cell Sorter (FACSCalibur). Only sodium azide and ammonium choride increased lysosomal pH, as revealed by an increase in FITC fluorescence. The most pronounced effect was obtained in the presence of both sodium azide and NH4Cl. (B) Calibration curves of FITC and Oregon green fluorescence in Dictyostelium endosomes. Dictyostelium cells were allowed to internalize either FITC-dextran or Oregon green-dextran for 20 minutes. The cells were then washed and transferred to HL5 medium containing 0.1% sodium azide and 40 mM NH4Cl, at the indicated pH. The fluorescence was measured by flow cytometry. The approximate pKa values (5 for Oregon green and 6.5 for FITC) are close to the expected values, confirming that the external pH is effectively imposed in endosomal compartments in these conditions.