Endosomal pH measurement

Dendritic cells were pulsed with a mixture of 40 kDa dextran fluorescein (1 mg/ml) and 40 kDa dextran Alexa-647 (1 mg/ml) (Molecular Probes) for 10 min in a water bath at 37 °C and then extensively washed with cold PBS 1% BSA. The cells were resuspended in complete medium incubated at 37 °C (for “chasing”) for the indicated times and immediately placed on ice to stop the reaction. To determine the endocytic population, a control experiment was maintained at 4 °C during the pulse. Cells were washed with PBS 1% BSA, resuspended in 100 μl of FACS buffer, and analysed using FACS. An FL1/FL4 gate was used to select cells that had endocytosed the fluorescent probes. Values were compared with a standard curve obtained by resuspending the cells that had endocytosed dextran at fixed pH (ranging from pH 5 to 8) in buffer containing 0.1% Triton X-100 for 8 min. The cells were immediately analysed using FACS to determine the emission ratio of the two fluorescent probes at each pH.