Figure 1.

NOX2 assembly at the phagocytic cup in monocyte-derived dendritic cells. (A) Representative confocal micrograph of a cell pulsed for 5 min with Alexa Fluor 633-labeled zymosan (magenta in merge) and immunostained for gp91^phox (cyan), p67^phox (yellow), and DAPI (blue). Scale bar: 2 μm. Graph: fluorescence intensity cross-sections over zymosan particles for multiple phagosomes. The bold cross-sections are from the left-hand images (indicated with the dotted line). Twenty-one phagosomes from 3 donors were analyzed. (B) Representative confocal micrograph of cell pulsed with FITC-labeled zymosan (magenta in merge) and immunostained for anti-FITC (in absence of permeabilization; yellow), p67^phox (with permeabilization; cyan) and DAPI (blue). Yellow arrow head: phagocytic cup. White arrow head: closed phagosome. Note the localization of p67^phox at the phagocytic cup. Scale bar: 2 μm. Twenty-nine phagosomes from 15 cells and 3 donors were analyzed. (C) Same as (B), but for gp91^phox (cyan). Scale bar: 2 μm. Thirty phagosomes from 16 cells and 3 donors were analyzed. (D) Mean fluorescent intensity (MFI) of p67^phox in the phagosome compared to the cytosol after 5 min of zymosan stimulation (as shown in B). Three donors were analyzed, each dot represents a single cell. p < 0.0001; paired Student's t-test. (E) Same as panel (D), but now for gp91^phox as in panel (C). p < 0.0001; paired Student's t-test. (F) Representative live cell epi-fluorescence imaging of a cell pulsed with zymosan labeled with both Alexa Fluor 633 (magenta) and nitrotetrazolium blue chloride (NBT) (transmission channel). Oxidation of NBT results in formation of dark formazan crystals (yellow arrow heads). Scale bar: 4 μm. Video file in Supplementary Movie 1. (G) Quantification of NBT crystal formation before and 25 min after attachment of a zymosan particle for six different donors (see also Supplementary Data 1). p = 0.009; paired Student's t-test. ^**p < 0.01; ^***p < 0.001.