DC Endocytosis of SVLP Figure 4. SVLP associating with early endosomes and CD9. (A) Pre-chilled DC were incubated with SVLP and CTB as in Fig. 3, or with antibody against CD-9 for 30 min on ice. Samples were then washed and shifted to 39uC for 2 hrs, followed by confocal microscopy analysis. High-resolution stacks prepared using IMARIS are shown for each label, arrows indicating co-localisation. Scale bars: 20 mm (left image), 5 mm (zooms). (B) DC were treated with 20 mM MBCD for 20 min at 39uC, followed by washing to remove the MBCD and pre-chilling for 30 min on ice. DC were then pulsed with 2.5 mg/ml SVLP for 20 min on ice (‘‘0’’ time point) and then washed with pre-chilled medium, prior to receiving fresh warm (39uC) medium containing 2 mM MBCD and shifting back to 39uC. DC were treated or not with PK to define internalisation at the time points shown. Analyses were by flow cytometry; all differences at the 39uC time points between 0 MBCD and 20 mM MBCD treatment are significant, as are the differences between ‘‘No PK’’ and ‘‘PK-treated’’ for the 20 mM MBCD samples (p#0.001). (C) DC were incubated with 2.5 mg/ml SVLP at 39uC for 10 min, then fixed/permeabilised and co-labelled with anti-EEA-1 antibody prior to confocal microscopy analysis. Scale bar: 3 mm. (D) A 3-dimensional analysis of the circle shown in (C); the right image shows a further analysis of the circle in the left image, analysed with the aid of Spots module in IMARIS to define centre points for SVLP (green) and EEA-1 (red) labelling. Scale bars: 3 mm (blend), 1 mm (zoom; spot module to enhance visualisation, filtered on mean intensity). doi:10.1371/journal.pone.0043248.g004 PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43248