DC Endocytosis of SVLP Figure 2. Internalisation of SVLP by DC. (A) DC incubated with 2.5 mg/ml SVLP (green) for 30 min at 39uC. High resolution stacks (102461024 voxels) were prepared, and 3D-imaging preformed using IMARIS. Scale bar: 5 mm. (B) SVLP (1 mg/ml) were added to pre-chilled DC (SVLP) or not (Mock) for 30 min on ice; pre-chilled DC were treated with PK before (DC + PK then SVLP) or after (DC + SVLP then PK) incubation with SVLP on ice. PK was added for 30 min on ice, then 10% serum added to ‘‘neutralise’’ PK activity. Cells were analysed by flow cytometry. The % Alexa488 positive cells were significantly different between SVLP (cells not treated with PK; ‘‘SVLP (positive control)’’) and ‘‘DC + PK then SVLP’’ (p = 0.006). (C) SVLP (1 mg/ml) were first pre-treated with PK as in (B). The treated SVLP were added to DC on ice for 30 min. Cells were washed and analysed by flow cytometry. (D) Pre-chilled DC were pulsed with 1 mg/ml SVLP for 20 min on ice and then shifted to 39uC for different time points, followed by treating or not with PK for 30 min on ice, and analysed by flow cytometry. (E) DC were incubated with different concentrations of SVLP for 24, 48 or 72 hrs at 39uC. Following washing, the cells were treated with PI and analysed by flow cytometry. The ‘‘0’’ on the x-axis indicates the cell control untreated with SVLP. (F) DC were incubated with 1 mg/ml SVLP for 20 min on ice, then different incubation time at 39uC, followed by flow cytometry analysis. Results are means of three samples 6 s.d. doi:10.1371/journal.pone.0043248.g002 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43248