stained with the former two antibodies were treated with 3mM NaN3 to impair receptor recycling. Antibodies and markers Considering the difficulties in expressing tagged proteins in primary DC using transfected DNA plasmids, visualisation of different cell organelles had to rely on direct labelling in situ. Accordingly, labelling of different intra-cellular organelles em- ployed the following reagents: Major Histocompatability Complex II (MHCII) containing vesicles were labelled with antibody against MHCII molecules (mouse monoclonal 1F12 IgG2b; BD Trans- duction Laboratories), microtubules were labelled with anti a- tubulin (mouse monoclonal IgM; Santa Cruz Biotechnology); early endosomes were labelled with antibody against Early Endosome Antigen 1 (EEA-1) (Mouse Anti-EEA-1; BD Transduction Laboratories); clathrin coated pits were labelled with anti-clathrin antibody (mouse anti-clathrin Heavy Chain; BD Transduction Laboratories); Endoplasmic Reticulum (ER) was labelled with antibody against Calnexin (pAB Rabbit; Enzo Life Sciences); lipid rafts were labelled with antibody against CD9 (Serotech) or cholera toxin B (CTB)-Alexa546 (Life Technologies, Zug, Switzer- land); CTB was used at a final concentration of 5 mg/ml. Isotypespecific conjugates labelled with Alexa-fluro dye were used from Life Technologies. Markers used for defining different endocytic pathways included biotinylated high molecular weight dextran-500 KDa (Sigma) for macropinocytosis, transferrin from human serum labelled with Alexa546 for clathrin-mediated endocytosis and recycling endosomes, CTB-Alexa546 for marking lipid rafts, DQ-Ovalbumin (DQ-Ova) to mark acidifying compart- ments, and ovalbumin labelled with Alexa488 for multiple endocytic routes; all from Life Technologies. Immunolabelling Four day-old DC (0.2 million cells/well) were seeded in 8-well fibronectin-coated Labteks (Nunc, Wiesbaden, Germany) with 200 ml of DMEM per well. For labelling of intracellular organelles, DC were fixed with 4% (w/v) paraformaldehyde (PFA) and washed twice with 0.1% (w/v) saponin, followed by incubation with the primary antibody (see above) diluted in 0.3% (w/v) saponin for 30 min on ice. Cells were then washed twice with 0.1% (w/v) saponin, followed by addition of an Alexa fluor- labelled conjugated secondary antibody diluted in 0.3% (w/v) saponin, with 30 min incubation on ice. Cells were then washed twice with 0.1% (w/v) saponin in cell wash. DC were then mounted in Mowiol and analysed by confocal microscopy. The exception to the above routine was the procedure to label microtubules. Cells were washed twice with microtubule stabilising buffer (MSB), and then permeabilised with 1% (w/v) Triton X- 100 in MSB for 1 min at room temperature. The cells were then washed and stained with the antibody against a-tubulin in cell wash for 30 min on ice, washed twice, and then given the Alexa fluor-labelled conjugated secondary antibody in cell wash for another 30 min on ice. After final washing, the cells were mounted in Mowiol and analysed by confocal microscopy. Confocal Microscopy Confocal microscopy employed a Leica TCS SL microscope and LCS software (Leica Microsystems AG, Glattbrugg, Switzer- land). All images were acquired using a 636 oil-immersion objective; analysis used IMARIS-7.4.2 software (Bitplane AG). For co-localisation analysis, high-resolution images acquired at opti- mum voxel size were used, and automatic threshold applied. Percentage material co-localised and Pearson’s coefficient were also calculated. All microscopy analyses employed threshold DC Endocytosis of SVLP subtraction and gamma-correction, relating to the negative controls that no false positive emissions were present. Some images had analyses with filament tracer and spot modules of IMARIS, to enhance their visualisation. DC Maturation Four day-old DC were treated with interferon-a (1000 U/ml) (in house) and lipopolysaccharide (LPS) (10 mg/ml) (Sigma) for 24 hrs at 39uC. Cells were then harvested and pulsed with SVLP on ice for 20 min., followed by washing and incubation at 39uC for different time points. Cells were then treated with PK or were directly analysed by flow cytometry. High molecular weight dextran and CTB-Alexa546 markers Cells were incubated with biotinylated high molecular weight dextran (50 mg/ml) for 30 min at 39uC, followed by washing and incubating on ice for 30 min. SVLP (2.5 mg/ml) were then added and further incubated on ice for 20 min. Cells were then washed and shifted to 39uC for different time points. DC were fixed with 4% (w/v) PFA at different times. Cells were then permeabilised (as above) and labelled with streptavidin-546 (Invitrogen) diluted in 0.3% (w/v) saponin. Four-day old DC were pre-incubated on ice for 30 min, followed by addition of SVLP, or CTB-Alexa546, or anti-CD9 antibody, or all together. The cells were then further incubated on ice for 30 min and then washed twice with cold PBS. Warm DMEM was then added and cells were shifted to 39uC for different incubation times, followed by fixing and mounting (as above). DC treatment with inhibitors DC were treated with 20 mM methyl-b cyclodextran (MBCD; Sigma) for 20 min at 39uC. Cells were then shifted on ice and incubated for 30 min. Then fresh medium with 2 mM MBCD was added to prevent cholesterol restoration, followed by addition of SVLP, with incubation for 20 min on ice. Cells were then washed and fixed with 4% (w/v) PFA, or stained labelled streptavidin prior to fixation. One set of samples were treated with PK and the other not. Flow Cytometry FACS Calibur analytical FCM (Becton Dickinson, Basel, Switzerland) was used for acquiring data, and CellQuest Pro software (Becton Dickinson) for analysis. Always 10,000 gated live cells were acquired. Statistical analysis employed the FlowJo software (Treestar, San Carlos, CA), which was also used to prepare the flow cytometry plots. Statistical Analysis For statistical analysis, the Student t-test was performed, using SigmaPlot Versin 11.0. Differences were considered to be significant when p#0.05. Supporting Information Figure S1 SVLP structure and PBMC sorting. (A) Structure of SVLP construct. (B) Purity of cell fractions isolated from PBMC by magnetic sorting using CD172a to isolate monocytes. (TIF) Figure S2 Influence of MBCD on SVLP and CTB interaction with DC. (A) Flow Cytometry histograms showing the uptake of SVLP by DC, triplicate samples, in the absence of PLOS ONE | www.plosone.org 15 August 2012 | Volume 7 | Issue 8 | e43248