Figure 1.

Phagocytosis of E. coli and activation of moDC.

(a) Flow cytometric gating strategy for moDC. Cells were first selected on forward and side scatter properties, followed by selection of single cells (FSC-H vs FSC-A). Subsequently, viable single cells were identified by exclusion of 7-AAD. Finally, moDC were selected based on cell surface expression of CD11c. (b-e) moDC were incubated in the absence (control) or presence (4, 8 or 24hours) of AF405 labelled E. coli (10 bacteria per moDC), harvested, stained with antibodies, and analysed by flow cytometry as in A. n=3 with each experiment indicated by separate symbols and mean values indicated with horizontal lines. (b) Viability of moDC was unaffected by incubation with E. coli. (c) All moDC expressed HLA-DR on their surface, irrespective of activation by E. coli. The percentage of activated moDC, as determined by expression of CD86, steadily increased to 95% during 24 hour of incubation with E. coli. The percentage of moDC that contained E. coli increased to 50% during the first 8 hour of incubation, and did not increase further up to 24 hours. Representative flow cytometry plots acquired after 24-hour incubation are shown in supplementary figure 2. (d and e) Geometric mean of HLA-DR and CD86 cell surface expression after incubation with E. coli expressed as fold increase relative to control. (f) Percentage of E. coli labelled moDC after 24 incubation with either 5 or 25 E. coli per moDC. n=3, with each experiment indicated by separate symbols and mean values indicated by horizontal lines.