American Society of Hematology

Figure 5

$Figure 5. Endosomal pH decreases in normal and CGD-DCs after dextran endocytosis. (A) Colocalization of FITC-dextran (green) and p47 (red) on endosomes of DCs, after a 10-minute endocytic pulse, as detected using confocal microscopy. (B) Endosomal ROS production measured by FACS. Control and DPI-treated DCs were pulsed with Oxyburst Green H2DCF coupled to BSA. Oxidation of the conjugate by endosomal ROS was detected by FACS. (C) DC endosomal pH kinetics after a 10-minute endocytosis pulse and the indicated time points of chase in the presence or absence of 10 μM DPI. The average and SD of 3 independent experiments are shown. Statistics were performed using the nonparametric Wilcoxon test for impaired data. **P < .01. (D) Control DC (blue) and CGD-DC (red) endosomal pH kinetics after a 10-minute endocytosis pulse and the indicated time points of chase. Statistics were performed using the nonparametric Wilcoxon test for paired data. *P < .05. (E) Endosomal membrane expression of Rab 5, lamp-1, gp91phox, and V-H+-ATPase after different time points of chase at 37°C following a 30-minute endocytic pulse at 4°C, as described in “Methods.” Purity of the fractions was assessed by evaluating the expression of gp96, Erk-2, and NF-κB.$

Endosomal pH decreases in normal and CGD-DCs after dextran endocytosis. (A) Colocalization of FITC-dextran (green) and p47 (red) on endosomes of DCs, after a 10-minute endocytic pulse, as detected using confocal microscopy. (B) Endosomal ROS production measured by FACS. Control and DPI-treated DCs were pulsed with Oxyburst Green H2DCF coupled to BSA. Oxidation of the conjugate by endosomal ROS was detected by FACS. (C) DC endosomal pH kinetics after a 10-minute endocytosis pulse and the indicated time points of chase in the presence or absence of 10 μM DPI. The average and SD of 3 independent experiments are shown. Statistics were performed using the nonparametric Wilcoxon test for impaired data. **P < .01. (D) Control DC (blue) and CGD-DC (red) endosomal pH kinetics after a 10-minute endocytosis pulse and the indicated time points of chase. Statistics were performed using the nonparametric Wilcoxon test for paired data. *P < .05. (E) Endosomal membrane expression of Rab 5, lamp-1, gp91phox, and V-H⁺-ATPase after different time points of chase at 37°C following a 30-minute endocytic pulse at 4°C, as described in “Methods.” Purity of the fractions was assessed by evaluating the expression of gp96, Erk-2, and NF-κB.

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