Dose-dependent effect of GNPs on death of DCs.

<p>(A) Necrosis and (B) apoptosis of DCs after 48 h cultures is shown from one representative experiment, or as mean ± SD (n = 4 experiments), after PI staining. (C) Different stages of DCs apoptosis were analyzed after 24 h cultures by annexin V:FITC/PI staining, and the results are presented from one representative experiment, or as mean ± SD of three independent experiments. *p<0.05 compared to control (Friedman's one-way ANOVA).</p

Physicochemical properties of GNPs in water and medium.

<p>*- Claimed by the vendor.</p><p>The concentration of Au within the GNP stock solutions was determined by Agilent Technologies 7500 ce ICP-MS, and it corresponded to the concentrations of Au reported by the vendor. The samples of GNPs (20 µg/ml) were prepared in DI 18 MEΩ water or complete medium, and sonicated for 20 s before the analysis by TEM or dynamic light scattering (DLS). The core size was analyzed on the preparations of GNPs added dropwise on the Cu-based grids and dried at room temperature, before the observation by JOEL JEM-2100 HR TEM (Tokyo, Japan) at 100 kV. The core size was similar in water and medium, so the average value from the measurements of GNPs in both solutions is shown as mean ± SD. Hydrodynamic size, polydispersion index (PDI), and z-potential in water and medium were analyzed by Zetasizer Nano ZS with non-invasive backscatter optics (NIBS). The samples were pre-warmed to 25°C for 120 s, and then analyzed for 15 min per run, for a total of 4 runs per sample, and the results are shown as mean ± SD (4).</p

Mechanisms of GNPs internalization by DCs.

<p>The internalization was analyzed by (A) SEM, under different magnifications; white arrows point to GNPs-membrane interactions; (B) Confocal microscopy of live cells stained with FM4-64; or (C) FIB/SEM using BSE, or SE detectors where indicated. White arrow points to the GNP₁₀-loaded vesicle attached to the outer membrane, whereas black arrows point to intracellular presence of GNP₁₀ agglomerates and a single particle of GNP₅₀. The cells shown in (B) and (C) were cultivated with GNPs for 3 h in the presence or absence of Dynasore; (D) TEM, after 24 h-cultures under different magnifications. Back arrows indicate intracellular GNP₁₀ agglomerates and a single GNP₅₀ particle outside the endosome.</p

Internalization of GNPs by DCs.

<p>Intracellular GNPs were analyzed in 48-cultures (A) after staining with MGG, (B) by flow cytometry, or (C) confocal microscopy. (D) Micro-PIXE analysis was used for the quantification of potassium (red) and gold (green) after 4 h cultures. Representative elemental maps are shown. (E) All data from micro-PIXE analysis is presented either as the amount of gold [pg/ml] per cell, or as the number of GNPs per cell, calculated from GNPs' size and density of Au (ρ = 19.3 g/cm³). *p<0.05 (Mann-Whitney test).</p

Effect of GNPs on Ca²⁺ oscillations in DCs.

<p>Fluo-3 loaded immature DCs were detected (A) immediately upon staining in the presence or absence of thapsigagrin (2 µM), or (B) after 24 h or 48 h in the presence of LPS and GNPs (10 µg/ml), as indicated. Representative kymographs and corresponding ΔFt/F₀ records are shown. The frequency of oscillations and, (C) area under peaks, are presented as mean ± SD of all analyzed cells. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096584#pone.0096584.s002" target="_blank">Figure S2</a>. *p<0.05, compared to control or as indicated; ^#p<0.05 compared to LPS; ⁺p<0.05 compared to GNP₁₀ (Kruskal-Wallis test).</p

Effect of GNPs on necrotic HEp-2-induced maturation and functions of DCs.

<p>(A) Phenotypic maturation of DCs is shown, as mean ± SE (n = 4 experiments); see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096584#pone.0096584.s001" target="_blank">Figure S1</a>; MFI-mean fluorescence intensity (B) DCs' capacity to stimulate the proliferation of allogeneic CD3⁺T cells, at 3 different DC/CD3⁺T ratios (X-axis), is shown as mean CPM ± SD of six replicates, from one representative experiment. (C) DCs' ability to produce IL-12p70, IL-23 and IL-10 after 48 h; (D) DCs' ability to induce production of cytokines by CD3⁺T cells in co-culture. (C) and (D) are presented as mean indexes ± SD (n = 4 experiments). (E) Metabolic activity of live HEp-2 cells co-cultivated with CD3⁺T cells, previously primed with DCs for 5 d. Results from a representative MTT assay are shown as mean ± SD of six replicates *p<0.05 (Friedman's two-way ANOVA).</p

Effect of GNPs on LPS-induced maturation and functions of DCs.

<p>(A) Phenotypic maturation of DCs is shown as mean ± SE (n = 4 experiments); see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096584#pone.0096584.s001" target="_blank">Figure S1</a>; MFI-mean fluorescence intensity (B) DCs' capacity to stimulate the proliferation of allogeneic CD3⁺T cells, at 3 different DC/CD3⁺T ratios (X-axis), is shown as mean CPM ± SD of six replicates, from one representative experiment. (C) DCs' ability to produce IL-12p70, IL-23 and IL-10 after 48 h; (D) DCs' ability to induce production of cytokines by CD3⁺T cells in co-culture. (C) and (D) are presented as mean indexes ± SD (n = 4 experiments). *p<0.05 (Friedman's two-way ANOVA).</p

Demographic characteristics of healthy volunteers who provided PBMCs.

<p>The blood was collected at the Institute of Blood Transfusion and Hemobiology of the Military Medical Academy, Belgrade, Serbia.</p