DC Endocytosis of SVLP Figure 6. SVLP association with DC microtubules, leading edge and MHCII. (A) Pre-chilled DC were incubated with 2.5 mg/ml SVLP for 20 min on ice, followed by shifting to 39uC for 10 min. Samples were then fixed, permeabilised and labelled with antibody targeting a-tubulin (red) and MHCII (blue). Acquisition was by confocal microscopy; high-resolution stacks were prepared and analysed using Filament Tracer and Spots modules in IMARIS. Visualisation was enhanced using filament tracer (microtubules) and spot (SVLP, MHCII) modules. Scale bars: 5 mm (left), 2 mm (right). (B) Pre-chilled DC were incubated with 2.5 mg/ml SVLP for 30 min on ice, followed by washing and shifting to 39uC for 20 min. Cells were then fixed, permeabilised and labelled with antibody targeting MHCII (blue). Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. Scale bar: 5 mm. (C) The same cultures were used as in (B), but incubated for 2 hours. Cells were then fixed, permeabilised and labelled with antibody targeting EEA-1 (red) or MHCII (blue). The arrows indicate sites of potential SVLP and MHCII association. Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. The arrow shows a vesicle in which SVLP and MHCII were present. Scale bar: 5 mm. doi:10.1371/journal.pone.0043248.g006 partially reduced in mature DC compared to immature DC (Fig. 8C), reflecting its known uptake by macropinocytosis plus other endocytic routes. Internalisation of SVLP (to become PK- resistant) by mature DC was also significantly reduced, whereas immature DC were 60% positive for SVLP within 5 min (Fig. 8D). Although these results indicate a major role for macropinocytosis in DC internalisation of SVLP, this is not the only endocytic process involved. Mature DC did internalise SVLP, but to a lower level and with a much slower kinetics. Discussion DC express a wide variety of receptors to constitutively sample their environment, and employ different endocytic pathways to PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43248