Fig. 3.

Kinetics of appearance of EαGFP and pMHC positive cells in lymph nodes draining the injection site. Flow cytometry was used to assess the proportion of CD11c^high and CD11c^low/− cells carrying EαGFP (A and B) and displaying pMHC complexes (C–F) at 1 h, 4 h, 12 h, 24 h and 48 h after EαGFP/LPS injection. Cell suspensions were prepared from the CLNs and BLNs of mice receiving either 100 μg EαGFP/LPS or PBS/LPS alone. Live cells were gated for CD11c expression and CD11c^high and CD11c^low/− gated cells were analysed separately for both GFP and Y-Ae staining. The mean percentage of CD11c⁺ cells in CLNs (closed circles, ●) and BLNs (closed triangles, ▴), that are GFP⁺ at each timepoint, are shown in (A). Open circles (○) and open triangles (▵) represent background staining in CLNs and BLNs of control mice receiving PBS/LPS only. Results for CD11c^low/− cells are shown in (B). The mean percentage of CD11c⁺ cells in CLNs, at each timepoint, that are surface Y-Ae⁺ following EαGFP (●) or PBS/LPS control immunisation (○) are shown in (C). Mouse IgG2b was used as the Y-Ae isotype control to set positive and negative gates and is represented as open symbols in (C). (D) Results for Y-Ae staining in CD11c^low/− cells. Kinetic results for CD11c^high and CD11c^low/− cells in BLN are shown in (E) and (F), respectively. Statistical comparisons were made between the EαGFP/LPS group (n = 3) and control PBS/LPS group (n = 3) for each timepoint and asterisk (*) indicates significance where p < 0.05 using the Student's t-test. Error bars show standard error of the means (SEM).