<p>(<b>a</b>) Left panel: flow cytometry dot plots of dLN cell suspensions to identify APCs are reported for each time point and each treatment (as indicated). Macrophages (MΦ) are depicted in blue, whereas the rest of the APCs are in black. The transient appearance of a CD8α_F4/80 double positive APC subset (red gates) between 8 hrs and 18 hrs after immunization (red quadrant) is shown. Representative results of one experiment out of three are shown. Right panel: bar graph histogram that reports the number x10<sup>3</sup> (per million of live/singlet cells acquired by flow cytometry) of CD8α_F4/80 double positive APCs at 8 hrs and 18 hrs after immunization with PE (white bars) and PE + MF59 (grey bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. **<i>P</i><0.01. (<b>b</b>) Flow cytometry dot plots of dLN cell suspensions obtained 18 hrs after immunization with PE or PE + MF59 are shown to analyze the expression of the surface protein CD64 on the different APC subsets. CD8α_F4/80 double positive APCs are depicted red, LN DCs in green and MΦs in blue. Representative results of one experiment out of the three independent experiments reported in panel (a) are shown. (<b>c</b>) Flow cytometry dot plots of dLN cell suspensions obtained 8 hrs and 18 hrs after immunization with MF59 reveal that the CD8α_F4/80 double positive APC subset (red cells) expresses the CD11c DC marker 18 hrs but not 8 hrs after the immunization. Representative results of one experiment out of the three reported in panel (a) are shown. (<b>d</b>) Flow cytometry dot plots of the dLN cell suspensions to identify APCs derived from mice immunized with MF59, whose LNs were explanted from both legs 8 hrs after the immunization, but: the LNs from the left legs (left dot plots) were immediately processed (8 hrs), whereas the LNs from the right legs (right dot plots) were processed after 10 hrs of in vitro culture (8 hrs + 10 hrs (in culture)). Representative results of one experiment out of three are shown.</p
