DC Endocytosis of SVLP Figure 1. SVLP binding to DC. (A) Graphic representation of SVLP. (B) DC were incubated with different concentrations of SVLP in medium without serum on ice for 20 min. Samples were washed and analysed by flow cytometry. (C) Pre-chilled DC were incubated with 2.5 mg/ml for SVLP for 5 or 30 seconds, then washed and fixed (10min, room temperature, 4% w/v p-formaldehyde) prior to analysing by confocal microscopy. Scale bars: 5 mm. (D) DC were incubated with high molecular weight dextran-546 (500 kDa), Ovalbumin-488, transferrin-546 (artificially coloured blue to aid visualisation) or PBS (mock) for 1 min at 39uC followed by analysis using confocal microscopy. Scale bars: 5 mm. (E) Pre-chilled DC were incubated with Ovalbumin-488 or PBS for 10 or 60 min on ice, followed by washing and analysis by flow cytometry. (F) Receptor-mediated binding of SVLP. DC were treated with pronase for 25 min at 39uC. Cells were then pre-cooled on ice for 30 min and washed 5 times. SVLP (1 mg/ml), or antibody against MHCI, CD172a or CD14, were added for 20 min on ice (antibody binding was then detected with Alexa488-conjugated anti mouse immunoglobulin F(ab’)2). The cells were also treated with 3 mM NaN3 to impair recycling of CD172a or CD14. Cells were analysed by flow cytometry. For the % SVLP positive cells, difference was significant between cells treated with pronase at ‘‘0.5 mg/ml’’ and ‘‘1 mg/ml’’ (p = 0.004), and also between ‘‘no pronase’’ and ‘‘0.5 mg/ml pronase’’ (p = 0.014). Results (B, E–F) are means of three samples 6 s.d. doi:10.1371/journal.pone.0043248.g001 PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43248