DC Endocytosis of SVLP Figure 8. SVLP association with high molecular weight dextran and its uptake by mature or immature DC. (A) DC were incubated with 50 mg/ml high molecular weight dextran for 30 min at 39uC, followed by ‘‘on ice’’ for 30 min. Cells were then treated with 2.5 mg/ml SVLP and incubated for 30 min, then shifted to 39uC for different times. Acquisition was by confocal microscopy; high-resolution stacks were prepared using IMARIS. White arrows: SVLP near the leading edge. Yellow arrows: Dextran+ vesicles in apposition with SVLP-vesicles. Scale bars: 50 mm (top-left), 3 mm (zooms). (B) Algorithms of SVLP co-localisation with dextran. (C) Four-day old DC were treated or not with interferon-a (1000 U/ml) and lipopolysaccharide (10 mg/ml) for 24 hrs to mature. High molecular weight dextran (50 mg/ml) or ovalbumin (10 mg/ml) was added, and incubated for 5, 30 or 60 min at 39uC. The number of positive cells was measured by flow cytometry; each bar represents means of three values and standard deviation. (D) Internalisation of SVLP (1 mg/ml) by the immature and mature DC used in (C). SVLP were added for 30 min on ice, then shifted to 39uC for different times, when the cultures were chilled and treated or not for 30 min with PK on ice. The number of positive cells was measured by flow cytometry; each bar represents means of three values and standard deviation. doi:10.1371/journal.pone.0043248.g008 PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e43248